WO1993016095A1 - Oligothionucleotides - Google Patents

Abstract

Chemical compounds consisting of an alpha or beta oligo-4'-thioribonucleotide or oligo-4'-thio-2'deoxyribonucleotide, characterized in that they comprise a concatenation of 4'-thioribonucleotides or 4'-thio-2'deoxyribonucleotides respectively, said concatenation being optionally linked to an effector, in particular a radical corresponding to an intercalating agent or a photoactivatable or chemical radical, e.g. a radical carrying a function which reacts directly or indirectly with the nucleotide chains, or a radical whose presence enables easy and sensitive detection. Methods for preparing said compounds, and their uses in therapeutics, diagnostics and as laboratory reagents, are also described.

Description

 OLIGOTHIONUCLEOTIDES

The present invention relates to new chemical compounds, as well as their applications. The chemical compounds according to the present invention are oligonucleotide compounds constituted at least in part by an oligo 4'-thio-ribonucleotide or an oligo 4'-thio-2 'deoxyribonucleotide comprising a chain of 4'thionucleotides.

 The compounds according to the invention can have the natural anomeric beta configuration or the unnatural anomeric configuration alpha.

 Thus are represented by the formulas (a) and (b) respectively of the 4'-thionucleotides alpha (a) and beta (b)

 (a) and (b) represent 4'-thionucleosides, it is necessary to add a phosphate at 5 'to obtain nucleotides.

 Under these conditions, multiple uses with biological and even pharmacological purposes already known for the oligonucleotides can be envisaged and this with more efficiency.

More specifically, the present invention relates to chemical compounds constructed by an oligo 4'-thioribonucleotide or an oligo-4'-thio-2 'deoxyribonucleotide, characterized in that they comprise a sequence of 4'-thioribonucleotides or 4 '-thio-2' deoxyribonucleotides respectively, chain which can be optionally linked to an effector agent, in particular, a radical corresponding to an agent of intercalation or a chemical or photoactivable radical, such as a radical carrying a function reacting directly or indirectly with the nucleotide chains or a radical whose presence allows an easy and sensitive detection.

 In particular, the subject of the invention is the new oligo-4'-thionucleotide derivatives, their preparation and their use in particular as probes allowing the detection of a defined sequence of nucleic acids, as artificial nucleases specific for DNA sequences or RNA or as selective biocage agents for the expression of a gene whether it is endogenous (for example, oncogenic gene) or exogenous (for example DNA or RNA of viruses, parasites or bacteria).

 In the French patent applications FR 8301223 (2 540 122) and FR 841 1795 (2 568 254), chemical compounds have been described consisting of an oligonucleotide or an oligodeoxynucleotide comprising a sequence of natural or modified nucleotides, that is to say say beta-nucleotides, to which is attached by a covalent bond at least one intercalating group, which have the property of selectively blocking the expression of a gene and which, therefore, are particularly useful in therapy as antiviral substances , antibiotics, antiparasitics or anti-tumor.

 In international PCT application WO 83/01451, a method has been described for blocking the translation of messenger TARN (mRNA) into protein by hybridization of the mRNA with an oligonucleotide having the sequence complementary to the mRNA, the oligonucleotide being stabilized in phosphotriester form.

 In international application WO 88/04301, oligonucleotides of alpha anomeric configuration have been described having parallel pairings with complementary sequences.

 Chemotherapy with antisense oligonucleotides concerns RNA or DNA targets of all living organisms (cells, bacteria, parasites, viruses or oncogenes).

The use of synthetic antisense oligonucleotides having the same structure as natural nucleic acids mainly encounters problems of nuclease sensitivity and cell penetration in a biological medium. To overcome these limits, oligonucleotide analogs capable of being more resistant to nucleases and of better penetrating into cells through the cytopiasmic membrane have been synthesized.

 It has in fact been described in the prior art derivatives of oligonucleotide compounds better resistant to enzymatic degradations in which the phosphate part was modified into thiophosphate or methyiphosphonate in particular. However, these derivatives have a chirality at the phosphate level capable of generating insoluble diastereoisomers.

 The present invention provides a new class of chimeric oligonucleotides, 4'-thio-oligonucleotides in which the intracyclic oxygen of the furanose cycle has been replaced by a sulfur atom.

 It has indeed now been found, and this is what is the subject of the present invention, that the substitution of oxygen by a sulfur on the sugar part of the nucleosides rather than on the phosphate group on the one hand, ensures the solubility of the oligomers and, on the other hand, could increase their penetration into the target cell, the substitution of oxygen by the sulfur element making the molecule more lipophilic.

 4'-thionucleotide derivatives form hybridization complexes with complementary RNA sequences much more stable than those formed with DNA and that, with respect to RNAs, 4'-thionucleotide derivatives form more stable hybridization complexes than natural beta-nucleotide derivatives.

 Among the compounds according to the present invention, there may be mentioned more particularly the beta oligomeric compounds of formula

in which : the β radicals may be the same or different and each represents a base of an optionally modified nucleic acid, activatable and / or comprising an intercalating group; the radicals X can be identical or different and each represents an oxoanion O -, a thioanion S -, an alkyl group, an aicoxy group, aryloxy, an aminoalkyl group, an aminoalkoxy group, a thioalkyl group, a substituted alkyl or aicoxy radical by a nitrogen heterocycle or a group -YZ;

 R and R ', which may be the same or different, each represents a hydrogen atom or a group -Y-Z or Y'-Z';

Y and Y ', identical or different, each represent a straight or branched alkylene radical -alk- or a radical chosen from,,,,, alk-O-alk

, and with U = O, S or N

 In particular, Y and Y 'represent a radical chosen from,,

with U = O, S or N

 E can have the same meanings as X except Y-Z or Y'-Z ';

 L represents O, S or -NH-;

 n and n 'represent an integer including 0;

 J represents a hydrogen atom or a hydroxy group;

Z and Z ', identical or different, each represents OH or a radical corresponding to an effector, in particular a radical corresponding to an intercalating agent or a radical carrying a function which reacts directly or indirectly with the nucleotide chains or a radical whose presence allows easy and sensitive detection.

 In this formula I, the following condensed representation of the nucleotides is used:

which corresponds to the structural formula

on which the ends (3 ') and (5') have been mentioned.

 It should be noted that formula I represents a sequence of 4′-thionucleotides which may be identical or different, of configuration D or L, n simply indicating the number of nucleotides included in the molecule; n is preferably a number between 1 and 50, and more preferably between 1 and 25.

 Mention is made in particular of the products for which L represents oxygen; R and R 'represent hydrogen and B a natural nucleic base, either adenine, thymine, cytosine or guanine when J = H and adenine, uracil, cytosine and guanine when J = OH.

Intercalating agents are products known in the art relating to nucleic acids; they are compounds capable to "interpose" in the structure of DNAs or RNAs, that is to say capable of being inserted between the basic plateaus of nucleic acids.

 The intercalating agents can be chosen from polycychic compounds having a flat configuration such as acridine and its derivatives, furocourmarin and its derivatives, daunomycin and other anthracycline derivatives, 1, 10-phenanthroline, phenanthridine and its derivatives, proflavin, porphyrins, dipyrido [1,2-a: 3 ', 2'-d] imidazole derivatives, ellipticine or elhpticinium and their derivatives and diazapyrene and its derivatives.

 The reactive chemical radicals can be radicals which can react directly or indirectly with a chain of nucleotides to form a covalent bond or to chemically modify it, or to cut it. Preferably, these reactive chemical radicals can be activated, for example, chemically, biochemically or photochemically.

 The activatable reactive radicals can be chosen from ethylene-diametetraacetic acid, diethylene-triaminepentaacetic acid, porphyrins, 1, 10-phenanthroline, 4-azido acetophenone, ethylene-imine, beta- chioroethylamine, psoralen and their derivatives, and aromatic compounds that absorb near-ultraviolet or visible radiation or that can react chemically with nucleic constituents.

 More particularly, the chemically activatable radicals in the presence of metal ions, oxygen and a reducing agent (ethylene-diamine-tetraacetic acid, diethylene-tri-amine-pentaacetic acid, porphyrin, phenanthroline) induce the cleavage in sequences nucleic acids located in their vicinity.

By irradiation in the visible or near ultraviolet domain, it is possible to activate the derivatives which absorb these radiations and to carry out bridging reactions or photo-induced reactions (cleavage and modification of nucleic bases) of nucleic acids. on which is fixed the oligothionucleotide carrying the activatable group. Among the radicals Z and Z ′ may be more particularly cited:

 - radicals derived from ethyiene-diamine-tetra-aceric acid of formula:

- radicals derived from diethyiene-triamine-pentaacetic acid, - radicals derived from methylpyrroporphyrin of formula:

- radicals derived from phenanthroline of formula

- radicals derived from acridine

- radicals derived from proflavin

R representing an amino (NH ₂ ) or azido (N ₃ ) group - the radicals derived from biotin

- radicals derived from azido-4 acetophenone of formula:

The β radical may preferably be chosen from natural nucleic bases (thymine, adenine, cytosine, guanine, uracil), but it is possible to use modified nucleic bases. Mention may be made of 2-amino adenine and its derivatives substituted, for example, on the nitrogen atom N by an aminoalkylene radical or by an azidophenyl-alkylene radical, guanine substituted on the oxygen atom O ⁶ , for example, by a (w-alkylene) -9-acridine group, (w-aminoalkyl) -amino-8-adenine and its derivatives substituted on the amino radical at w by an acridine group, or the halogenated or azide derivatives such than bromo-5 uracil, azido-8 adenine, 7-deazaadenine, 7-deazaguanine. It is also possible to use derivatives of the nucleic bases comprising an intercalating group or a chemically or photochemically activatable group.

Thus, the functionalization of C or T by an aziridine group in position 4 leads to the formation of covalent bridges CH ₂ -CH ₂ between the two complementary strands on G and A respectively. Therefore, more particularly, the β radical is then chosen from 4-azido-cytosine or 4-azido-thymine.

Preferably, the radical X represents an oxoanion. However, the radical X can represent an alkyl radical containing 1 to 7 carbon atoms (methyl, ethyl, propyl), an aicoxy radical in which the alkyl part contains 1 to 7 carbon atoms (methoxy, ethoxy, dimethyl-2,2 propyloxy), an aminoalkyl or aminoalkoxy radical of general formula R ₁ R ₂ N-alk-A- in which A represents a bond or an oxygen atom, -alk- represents an alkylene radical containing 1 to .10 carbon atoms and R ₁ and R ₂ , identical or different, represent a hydrogen atom or an alkyl radical containing 1 to 7 carbon atoms or form together with the nitrogen atom to which they are linked a saturated nitrogen heterocycle with 5 or 6 members , it being understood that the group R ₁ R ₂ N-can be quaternized, or an alkylthio radical in which the alkyl part contains 1 to 7 carbon atoms.

In formula (I), the radical -alk- is preferably a straight or branched alkylene radical having from 1 to 10 carbon atoms. In particular, starting from the 3 ′ or 5 ′ alcohol terminal functions of the oligothionucleotide, the effector Z can therefore be introduced via a chain (CH ₂ ) _n linked to a functionalization Z ′, of various kinds at the osidic part of the oligomer.

 From the following formula:

according to what Z ^' ₁ represents, for example

- a phosphate or methyl phosphonate of formula

U = O, N or S

- an ether of general formula

- an ester of general formula:

or

- a carbamate of general formula:

In general, n 'is between 1 and 10. The present invention also relates to the above compounds in the form of a salt with bases or acids, and the compounds in racemic form, or in the form of purified or mixed optical R or S isometers, of formula (I).

 The present invention preferably relates to the preceding compounds of formula (I) in the series D.

 Mention is made in particular of the D-oligothionucleotide compound products of general formula:

in which J and X are defined as above for the compounds of formula (I) and ß represents a natural nucleic base.

The oligonucleotides according to the present invention can be in the form of homogeneous sequences, as in the compounds of formula (I) or in the form of mixed oligonucleotides, that is to say in the form of specific sequences included in other types DNA or RNA type oligonucleotides, modified or not at the phosphate chain, the latter corresponding to compounds of formula (I), in which the intracyclic oxygen of the. furanosis cycle is restored. The present invention therefore also relates to mixed oigomeric compounds characterized in that they consist of oligothionucleotide compounds according to the invention linked to oligonucleotides of DNA or RNA type. The term “DNA or RNA type oligonucleotides” is understood here to mean a DNA or RNA type nucleic acid sequence modified or not at the phosphate chain and in which the intracyclic heteroatom of the furanosis cycle of the nucleotides is oxygen.

 The subject of the present invention is in particular mixed oligomeric compounds constituted by an oligothionucleotide sequence according to the invention, comprising an oligonucleotide sequence of DNA or RNA type within it or at one of its ends.

 In a particular embodiment, therefore, the oligonucleotide sequence, of DNA or RNA type, is framed by two sequences of oligothioribonucleotides.

 The new products of general formula (I) or mixed oligomers containing them can be prepared chemically by known methods and, in particular, those which are described in the applications for known methods and, in particular, those which are described in French patent applications FR 8301223 (2 540 122) and FR 841 1795 (2 568 254), WO 88/04301 and FR 88 122648.

 The oligothionucleotide compounds in particular of formula (I) can be prepared chemically by methods in which syntheses are applied to the conventional phosphodiester, phosphotriester, phosphoramidite or hydrogenophosphonate for oligonucleotides.

 In these processes, the chain of

4'-thionucleotides, the different groups not used being protected, then the protective groups are finally eliminated to obtain the desired products.

 Mention is made in particular of a process for the synthesis of oligothionucleotide compounds according to the invention without an effector agent, characterized in that a supported synthesis is carried out according to the phosphoroamidite method comprising

protection at 3 'and 5' of the starting 4'-thionucleotides or oligo4'-thionucleotides with, for example, dimethoxytrityl at 5 'and methyl diisopropylaminophosphoramidite at 3', the functionalization of a solid support incorporating a 4'-thionucleoside derivative by a link, for example succinyl, between the 3'-hydroxyl group of the 4'-thonucleoside derivative and an amino group of the solid support,

- elongation of the oligothionucleotide chain in a synthesizer reactor,

 - finally the stall, deprotection and purification of the extended oligothionucleotide.

 There is also cited a process for the synthesis of oligothionucleotide compounds incorporating an effector agent according to the invention, characterized in that one starts from an oligothionucieotide without an effector, or from a mixed ohgomer comprising, protected in 5 ′, of which reacts the OH 3 ′ termination with an unprotected function of a difunctional arm whose second function is protected and after deprotection of the resulting product, said product is linked to the effector by the second free function of the difunctional arm.

 The OH 3 ′ end of the 5 ′ protected oligomer can be esterified with an aminohexanoic acid, the amino function of which is protected.

 A process for the solid phase preparation of oligothionucleotide sequences according to the phosphoramidite method, object of the present invention, comprises the following essential steps:

 - A 4'-thionucleoside derivative protected is immobilized by means of one of its hydroxyl functions in 3 'or, if appropriate, 2' on a solid support.

 - We assemble on this support in a manual or automatic synthesizer reactor the oligothionucleotide chain protected by condensation of monomers consisting of protected 4'-thionucleosιdes substituted by phosphoramidi tes groups in 3 ', where appropriate, the hydroxyl function in 2' riboses being protected by the Ctmp or TBDMS groups.

- The oligothionucleotides are obtained after detachment of the oligomer obtained from the support and elimination of the protective groups. Suitably, the oligothionucleotide is assembled by condensation, in the presence of an activating agent, of said monomers between their 3 'function and the 5' function of the 4'-thionucleoside compound immobilized for the first monomer or d 'a protected polythionucleotide intermediate compound attached to said immobilized thionucieoside compound for the following monomers.

 In particular, the activating agent for the condensation of said monomers can be chosen from tetrazole and its derivatives, such as para-nitrophenyl tetrazole.

 Advantageously, the phosphoramidite group at 3 ′ of said monomers is an N, N dialkylaminophosphoramidite group of formula

with R ₁ which represents an optionally substituted identical or different C ₁ to C ₇ alkyl, such as CH ₃ , (CH ₃ ) ₂ CH.

R ₂ which represents an optionally substituted C 1 -C 4 alkyl, such as CH ₃ , CH ₂ CH ₂ CN.

In particular, the phosphoramidite group at 3 ′ of said monomers is methyl dnsopropylaminophosphoramidite (R ₁ = (CH ₃ ) ₂ CH and R ₂ = CH,) or 2-cyanoethyl dnsopropylaminophosphite (R ₁ = (CH ₃ ) ₂ CH and R ₂ = CH ₂ CH ₂ CN).

 Suitably, the hydroxyl groups of said monomers and of said immobilized 4'-thionucleoside compound can be protected 5 'by the group DmTr.

 The immobilized 4'thionucleoside compound can be attached to the solid support via a divalent succinyl group between one of its OH groups in 2 'or 3' which is thus esterified, and an amino group of the support.

The solid support can consist of a long alkylamine chain attached to a polymer, silica gel or porous glass beads. Mention may be made, as functional group permitting attachment or being linked on a solid support endowed with an amino group, the group of formula

 with p = 1 to 5,

in which R ₄ represents H an activating group such as C ₆ -Cl ₅ or an NH radical linked to a solid support such as the NH radical of an alkylamine chain attached to a polymer, silica gel or glass beads, porous.

 An advantage of the homogeneous or mixed compounds according to the present invention, comprising a chain of oligo-thio-nucleotides, is also to be able to envisage their use independently of effector agents, in particular of intercalating agents, since these new oligonucleotide substances ensure recognition of the complementary nucleic acid sequence, in particular of RNA, with very increased stability; the function of the intercalating agent being to increase the stability of the hybridization complex, its presence is no longer mandatory.

 The compounds according to the invention, by their high specific affinity of - oligothionucieotide sequences for complementary nucleic sequences, are in fact superior to current oligonucleotides whose affinity for complementary sequences is weaker.

 The compounds according to the present invention can therefore be used more effectively, as hybridization probes, but also as purification components for specific DNA or RNA sequences.

 These compounds can also be used to detect mutations in DNA or RNA more efficiently.

The compounds of the present invention, by the property of the oligothionucieotide sequences to bind strongly to the complementary nucleic sequence, then to induce the cleavage of the chain of nucleic acids at this site, in particular when they are coupled to an agent for cut, can be used as sequence specific artificial nucleases. They can be used as reagents in molecular biology or in genetic engineering. The presence of the 2 'hydroxyl function, where appropriate, also makes it possible to envisage their use as artificial ribozymes, whether in the form of homogeneous or mixed oligothioribonucleotide sequences, that is to say associated with oligonucleotides of DNA or RNA type.

 The object of the present invention is also the application of the compounds according to the invention for carrying out the specific blocking of cellular genes or pathogenic agents previously chosen (viruses, bacteria, parasites).

 The compounds according to the invention are therefore also usable as medicaments.

 The new products of general formula (I) according to the invention and the products of general formula (I) in which L represents an oxygen atom, R and R 'each represent a hydrogen atom and B represents a natural nucleic base form hybridization complexes with complementary RNA sequences much more stable than those formed with DNA.

 In particular, the products of formula (I) in which J = OH (oligothioribonuciéotides) pair more stable with complementary RNA sequences than the products of formula (I) in which J = H (oligothiodoxyoxyonucleotides).

 Given the fact that the targets of the antisense molecules in therapies are the RNA messenger genes, this property of the oligothioribonuciéotides is quite interesting.

 This difference in stability of the hybridization complexes allows preferential inhibition of messenger RNAs and viral RNAs without any significant risk of causing undesirable effects in the DNA of the genome.

 In addition, with respect to RNAs, the products according to the invention generally form more stable complexes than those which are obtained from natural oligonucleotides.

The oligothioribonucionotide compounds according to the invention, in particular of formula (I) (J = OH), also exhibit a certain number of advantageous characteristics, in particular for application as an antisense agent: - the natural beta configuration is respected,

 - they are isoelectric from natural RNAs,

 - they have no chirality at the phosphodiester bond,

- they are more lipophilic than natural analogues because of the presence of the sulfur atom.

 The sequences of the oligothionucleotides, coupled or not coupled to an intercalating agent or to a chemically or photochemically activatable group, comprising a chain of pyrimidine nucleosides are capable of binding to a double helix of DNA or RNA comprising a sequence of adjacent purine bases associated by hydrogen bonding to the complementary sequence of the pyrimidine bases. This fixation involves the local formation of a triple helix in which the pyrimidine bases of the oligothionucleotide form hydrogen bonds (of Hoogsteen or reverse Hoogsteen type) with the purine bases of the double helix. In this context, the oligothionucleotides form more stable complexes than the corresponding oligonucleotides and they make it possible to induce irreversible reactions (bridging, modification or cleavage) on a double helix of RNA or DNA.

 The new products of general formula (I) according to the invention and the products of general formula (I) in which L represents an oxygen atom, R and R 'each represent a hydrogen atom and B represents a natural nucleic base , which have the property of binding strongly to the complementary nucleic sequence, or mixed oligomers including it, can be used as probes to detect the presence of a chain of complementary nucleotides. This detection is facilitated by the presence of a fluorescent group or a biotin group in these molecules.

 The unnatural structure of oligothionucleotides confers antigenicity properties thus making possible the preparation of specific antibodies directed against oligothionucleotides possibly coupled to an intercalating agent or against their complexes with a natural DNA or RNA.

For diagnostic or prognostic purposes, the oligothionucleotides, optionally coupled to an intercalating agent, or mixed oligomers including them, can be covalently attached to a solid support. Coupling to a luminescent marker or generator of a colored, luminescent or fluorescent reaction allows them to be used as DNA or RNA sequence probes for diagnostic or prognostic purposes.

 The oligothionucleotides according to the invention are much more resistant to nucleases than derivatives of natural nucleosides. This stability with respect to enzymatic hydrolysis allows the use of the products according to the invention, in particular of general formula (I), or mixed oigomers comprising, in in vivo or in vitro experiments in the presence of nucleases. Therefore, the oligothionucleotides according to the invention have indisputable advantages over the beta-nucleoside derivatives already known.

 The oligothioribonucleotide compounds of formula (I) for which J = OH are more resistant to enzymatic degradation than the oligothiodoxyoxyonucleotide compounds of formula I for which J = H.

 Consequently, as we have seen, another object of the present invention relates to the application of the new products according to the invention, in particular of general formula (I), and of products of general formula (I) in which L represents an oxygen atom, R and R 'each represent a hydrogen atom and ß represents a natural nucleic base, in particular the products for which J = OH, or mixed oligomers comprising, with specific biocage of cellular genes or pathogens previously selected (viruses, bacteria, parasites), in particular mRNAs.

The new products according to the invention, in particular of general formula (I), and the products of general formula (I) in which L represents an oxygen atom, R and R ′ each represent a hydrogen atom and ß represents a natural nucleic base, in particular the products for which J = OH, or mixed oligomers including them, are particularly useful as drugs making it possible to block undesirable exogenous genes (viruses, bacteria, parasites) or endogenous (cellular genes, oncogenes), in particular mRNAs. The expression of these genes can be blocked by acting either directly on the DNA or the RNA carrying the genetic information, or on the messenger RNA, copy of the gene, by then blocking any translation by hybridization or by hybridization then bridging or modification or cutting of the messenger or viral RNA chosen as target.

 This blocking can be achieved by using a product according to the invention, in particular of general formula (I), or a product of general formula (I) in which L represents an oxygen atom, R and R ′ each represent an atom d hydrogen and B represents a natural nucleic base, in particular the products for which J = OH, or mixed oligomers comprising them, the sequence of which is complementary to that of an uncomplexed region of an RNA or of a DNA and, in particular, a messenger RNA. Hybridization, whether or not followed by bridging, modification or cutting of messenger or viral RNA, prevents the synthesis of the RNA or the corresponding protein or the expression of viral or parasitic functions.

 If this RNA or this protein is vital for the virus, the bacteria or the parasite, the products according to the invention, in particular of general formula (I), and the products of general formula (I) in which L represents an atom of oxygen, R and R 'each represent a hydrogen atom and B represents a natural nucleic base, in particular the products for which J = OH, or mixed oigomers including them, will constitute drugs with antiviral, antibacterial or antiparasitic activity.

If this RNA or this protein is not vital for the organism, it is possible to selectively suppress its effects. In this case, the products according to the invention, in particular of general formula (I), and the products of general formula (I) in which L represents an oxygen atom, R and R ′ each represent a hydrogen atom and B represents a natural nucleic base, in particular the products for which J = OH, or mixed oligomers including it, will constitute either drugs with antitumor activity when the targeted gene or its messenger RNA codes for a protein involved in cell transformation, or drugs capable of suppressing resistance to antivirals, antibiotics or antiparasitics when the encoded protein is responsible for the inactivation of antibiotics, antivirals or antiparasitics. Specific cytotoxic effects can be obtained by the action of a product according to the invention, in particular of formula (I), or of a product of general formula (I) in which L represents an oxygen atom, R and R ' each represents a hydrogen atom and ß represents a natural base, in particular the products for which J = OH, or mixed oligomers including / understanding, on a cellular function essential to the target cell.

The mixed oligomers of igothioribonucleotides according to the invention, comprising a DNA oligonucleotide sequence where intracyclic oxygen is restored, are particularly advantageous in use as an antisense agent. Such mixed oligomers are likely to be highly selective to the extent that the "window" of DNA structure is one substrate for RNAse H after pairing with its complementary antisense RNA target sequence _m, said RNAse H inducing then cleavage of the DNA / RNA complex.

 Other characteristics and advantages of the present invention will become apparent in the light of the examples which follow.

Figure 1 represents the thermal stability curve of the betadT ₅ duplex (S _r T) dT ₆ / betadC ₂ A ₁₂ C ₂ compared to that of the natural duplex betadT _{1 2} / betadC ₂ A _{1 2} C ₂ .

Figure 2 represents the curve of enzymatic digestion of beta (S _r T) dT ₁₁ .

In the description which follows, the abbreviation rS or S precedes a thioribonucleotide.

I-SYNTHESIS OF THIONUCLEOTIDES.

I. 1- The first syntheses of thiosugars were carried out between 1960 and 1970.

 Two examples are given:

 The synthesis of 4-thio-D-ribofuranose derivatives is carried out according to scheme 1:

 This compound is obtained in 8 steps from L-Lyxose with an overall yield of 6%.

- R. L. WHISTLER, W. E. DICK, T. R. INGLE, R. M. ROWELL and B. URBAS. J. Org. Chem., 1964, 29, 3723-3725.

 - B. URBAS and R. L. WHISTLER, J. Org. Chem., 1966, 31, 813-816.

 - E. J. REIST, D. E. GUEFFROY and L. GOODMAN, J. Am. Chem. Soc., 1964, 86, 5658-5663.

 - R. L. WHISTLER and J. N. BeMILLER in "Methods in Carbohydrate Chemistry, R. L. WHISTLER and M. L. WOLFROM, Eds., Academie Press, Inc., New York, 1962, Vol.

I, p.79. The 4-thio-2-deoxy-D-ribofuranose derivatives were obtained according to scheme 2:

This 14-step synthesis leads to methyl 2-deoxy 4-thio-D-erythro-pentofurannoside with an overall yield of 8%

- Y. L. FU and M. BOBEK. J. Org. Chem. 1976, 41, 3831-3834.

- Y. L. FU and M. BOBEK, in "Nucleic Acid Chemistry". L. TOWSEND and R. S.

 TIPSON.Eds., 1978. Part I pp. 183-194

 - U. G. NAYAK and R.L. WHISTLER. Liebigs Ann Chem., 1970, 741, 131-138.

Similarly, derivatives of 4-thio-D-arabinofuranose were obtained

 - R. L. WHISTLER. U. G. NAYAK and A W PERKIN Jr., J. Org., Chem., 1970, 35, 519-521

I. 2. The corresponding nucleosides were then synthesized:

 a) In series 4'-thio-2'-deoxy-D-ribofurannose

- Y. L. FU and M. BOBEK in "Nucleic Aad Chemistry", L. TOWSEND. R. L. TIPSON. Eds., John Wiley & Sons. New York. 1978. p. 317 b) In series 4'-thio-D-ribofurannose

- E. J. REIST, D. E. GUEFFROY and L. GOODMAN. J. Am. Chem. Soc., 1964, 86, 5658-5663.

 - E. J. REIST, D. E. GUEFFROY and L. GOODMAN. Chem. Ind., 1964, 1364-1365

- B. URBAS and R. L. WHISTLER. J. Org Chem., 1966, 31, 813-816.

 - M. BOBEK, R. L. WHISTLER and A. BLOCH. J Med. Chem., 1970. 13, 41 1-413

- M. BOBEK, A. BLOCH, R. PARTHASARATHY and R. L. WHISTLER. J. Med. Chem., 1975, 18, 784-787.

 - M. BOBEK, R. L. WHISTLER and A. BLOCH. J. Med. Chem., 1972, 15, 168-171.

- N. OTOTANI and R. L. WHISTLER. J Med. Chem., 1974., 17. 535-537

 - Mr. W. PICKERING. J. T. WITKOWSKI and R. K. ROBBINS. J. Med. Chem., 1976, 19, 841-842.

 - A. K. M. ANISUZZAMAN and M. D. AMIN. J. Bangladesh Acad. Sci., 1978, 2, 59-64 - D. J. HOFFMAN and R. L. WHISTLER. Biochemtstry, 1970, 9, 2367 - 2370 c) In other series:

- R.G.S. RITCHIE and W. A. SZAREK. J Chem. Soc. Chem. Commun., 1973. 686.

- E.J. REIST. L. V. FISCHER and L. GOODMAN. J. Org. Chem., 1968, 33, 189. - R. L. WHISTLER. L. W. DΟNER and U. G. NAYAK. J. Org. Chem., 1971, 36, 108.

1 10 These nucleosides are obtained by the traditional routes of the synthesis of nucleosides in an oxygenated series.

Or by the heavy metal salt method:

Treatment of the chloromercuric heterocycle with the corresponding chlorosugar.

 - Mr. BOBEK. R. L. WHISTLER and A. BLOCH. J. Med. Chem., 1972, 15, 168-171

 - D. E. GUEFFROY and L. GOODMAN, Chem. & Ind., 1964, 1364-1365

 - E. J. REIST. Mr. BOBEK. R. L. WHISTLER and A. BLOCH. J. Med. Chem., 1970, 13, 41 1-413.

- E. J. REIST. D. E. GUEFFROY and L. GOODMAN. J. Am. Chem. Soc., 1964, 86, 5658-5663.

 - E. J. REIST. L. V. FISCHER and L. GOODMAN, J. Org. Chem., 1968, 33, 189-192

Either by the method of HILBERT and JOHNSON,

 - by condensation of the pyrimidine base with the corresponding thiochlorosugar.

 - B. URBAS and R. L. WHISTLER. J. Org. Chem., 1966, 31, 813-816.

 - by condensation of silylated derivatives of bases and of the corresponding thiochlorosugars

- Mr. BOBEK. A. BLOCH, R. PARTHASARATHY and R. L. WHISTLER. J. Med Chem., 1975, 18, 784-787.

 - R. L. WHISTLER. L. W. DONER and U. G. NAYAK. J. Org. Chem., 1971. 36, 108-1 10 - N. OTOTANI and R. L. WHISTLER. J. Med. Chem., 1974. 17, 535-537

 - By the fusion reaction of 3-cyano 1,2,4 triazole and 1,2,3,5 tetra-O-acety l-4-thio-D-riborurarmose.

 - M. V. PICKERING, J. T. WITKOWSKI and R. K. ROBINS. J. Med. Chem. , 1976, 19, 841-842.

 - by the method of VORBRUGGEN and NIEDBALLA by condensation of adenine and 1,2,3.5 tetra-O-acetyl thioribofuranose in the presence of FRIEDEL and CRAFTS catalyst.

 - A. K. M. ANISUZZAMAN and M D. AMIN, J. Bangladesh Acad. Sci., 1978, 2, 59-64

Recently, the synthesis of thionucleosides has again gained interest.

Thus, the 2 ', 3', 5 'tri-O-benzyl 4'-thio-ß-D-xylofurannosyl uracil was obtained by an original route according to scheme 3.

- Mr. W. BREDENKAMP. C. W. HOLZAPFEL and A. D. SWANEPOEL. Tetrahedron Lett. , 1990, 31, 2759-2762.

- MW BREDENKAMP. CW HOLZAPFEL and AD SWANEPOEL. S. Air J Chem. 1991. 44, 3.1-33.

(i = dibutyltin oxide; ii = MsCl; iii = BnCl: iv = tetra-butylammonium iodide. barium carbonate; v = Hg (OAc) ₂ / AcOH; vi = Modified HILBERT JOHNSON method.)

DIAGRAM 3

Union of 4'-thio 2'-deoxy nucleoside pyrimidine analogs has been published

- M. R. DYSON. P. L. COE and R. T. WALKER. J. Med. Chem., 1991, 34, 2782-2786. by the method of HORTON and MARKOVS

 - D. HORTON and R. A. MARKOVS, Carbohydrate Res., 1980, 80, 356.

It is illustrated by diagram 4:

 DIAGRAM 4

Similarly, 2'-deoxy-4'-thiocytidine. 2'-deoxy-4'-thiouriduie and 4'-thiothymidine were obtained by J. A. SECRIST III and Coll. (J. A SECRIST. K N TIWARI. J. M. R1ORDAN and J A MONTGOMERY. J Med. Chem. 1991, 34, 2361-2366). (JA MONTGOMERY and J A. SECRIST IIL PCT Int Appl WO 9104 033) from 2-deoxy-4-thio-ß-D-y-thro-pentofuranose synthesized according to BOBEK and Coll (YL FU. M BOBEK. J. Org. Chem. 1976, 41, 3831-3834.) Using TMSTf as a coupling catalyst

The synthesis of 4'-thio 2'-deoxy-D-ribofuranose has been the subject of a patent and publications

- European Patent Application. 0421 777 Al R. T WALKER

 - M. R. DYSON. P. L. COE and R T WALKER. J Chem Soc Chem. Comm., 1991,741- 742

- MR DYSON. P. L COE and RT WALKER. Carbohydrate Res., 1991, 216, 237-248 It is depicted in diagram 5: B

(i = HCl / MeOH. ii = BnBr / THF, iii = BnSH / HCl, iv = Ph ₃ P / DEAD / BzOH / THF. v =

MeONa / MeOH / CH ₂ Cl ₂ , vi = MsCl / Pyr., Vii = Nal / CO ₃ Ba / Acetone, viii = BCI ₃ /

CH ₂ CI ₂ , ix = p-toluoylCl, x = Ac ₂ O / H ₂ SO ₄ )

DIAGRAM 5 Ribothionucleosides derived from pyrimidines were the subject of the same patent. But the synthesis of the starting thioribofuranosis was carried out according to the method of EJ REIST - EJ REIST, DE GUEFFROY and L. GOODMAN. J. Am. Chem. Soc., 1964, 84, 5658 I. 3. SYNTHESIS OF SYNTHONS OF 4'-THIO-BETA-D-RIBOFURANNOSE

 NECESSARY TO OBTAIN TH1OOLIGOMERS.

The synthesis of L-thioLyxofurannose and D-thioRibofurannose was carried out in 7 steps from D-ribose and L-lyxose respectively with 29 and 21% yield respectively. This synthesis is illustrated in diagram 6

(i = MeOH, HCl: ii = BnBr, KOH; iii = HCL, H ₂ O, dioxane; iv = BnSH, HCl. v = MesCL.

pyr. "vi = NBu ₄ I. BaCO ₃ ; vii = Hg (OAc) ₂ )

FIGURE 6 The synthesis to obtain thio-D-ribofuranose uses L-lyxose as a starting product as in the synthesis of WHISTLER (page 19). This strategy is called strategy C1— C4. Indeed, the sulfur atom is first of all introduced in the anomeric position and a nucleophylic substitution SN2 between the sulfur atom carried by the anomeric carbon (C1) and the C4 carbon carrying an OH activated by a Mesyl group. (Ms) is then performed. This strategy applied to the synthesis of 4-thio-D-ribofurannose and 4-thio-L-lyxofurannose resembles that which WALKER used to achieve the 2-desoxy-D-ribofurannose series (Eur. Pat. 0421 777 A 1 page 6). For example, product 5 (Figure 5) from WALKER is similar to our product 10 (Figure 6) in the L-Lyxofurannose series.

 FIGURE 7

The synthesis of uracil duonucleosides, diymine and cytosine was carried out using bis-trimethylsilyl acetamide (BSA) as silylating agent and trimethylsilyl trifluoromethane sulfonate (TMSTf) as coupling agent. Nucleosides O and ß were obtained with 74, 77 and 70% yield respectively. The synthesis of adenine thionucieosides was carried out by SnC14 in acetomtrile. Nucleosides O and ß are obtained with 58% yield (Scheme 7).

II. SYNTHESIS OF OLIGO-THIONUCLEOTIDES ON SOLID SUPPORT. The supported synthesis of ß oligo-thioribonucleorides was carried out using a GENE synthesizer model APPLIED BIOSYSTEM 318A according to the phosphoramidite method.

 It is similar to that described for the ß series natural oligodeoxyribonucleotide by

CARUTHERS et al. (S. L. BEAUCAGE and M. H. CARUTHERS. Tétrahedron Utt.,

1981, 22, 1859-1862; L. J. Me BRIDE and M. H. CARUTHERS, Tétrahedron Leit., 1983, 24,

245-248.)

and to that described by USMAN and Coll. for oligoribonucleotides (N. USMAN. K K

OGILVIE. M. Y. JIANG and R. J. CEDERGREN. J. Am. Chem. Soc., 1987. 109. 7845- 7854.).

This synthesis required:

 I) The preparation of synthons ß thioribonucleoside phosphoramidites 5 corresponding to the bases T. U. C, A and G.

 II) Functionalization of the solid support incorporating a β thioribonucleoside derivative.

 III) The elongation of the oligothioribonucleotide chain using an automatic synthesizer.

 IV) Finally, at the end of the required number of synthesis cycles, the oligothionucleotide has been removed from its deprotected and purified support. II.1 - Synthesis of the phosphoramidite of beu-thioribonucleoside 26 a-d.

 II. 1. 1 Synthesis of 5'-DmTr-2'-O-TBDMS derivatives (Diagram 8)

The major problem in the chemical synthesis of ugoribonucleosides is the presence of 2 'hydroxyl in the ribose or thioribose cycle which requires selective protection

The selection of a protective group for the 2 'hydroxyl must correspond to several criteria, it must be stable:

 - under the coupling conditions during the deprotection of the protective groups in 5 'to allow the extension of the chain.

 - during oxidation in the case of the phosphite triester method

 - during the masking which follows the coupling.

 - during the deprotection of the protective groups of the exocychic amines

- during deprotection of phosphates.

- And in the case of syntheses on solid support during the cleavage of the oligomer from the support. Finally, this protective group must be removed under very mild conditions to avoid an attack on the 2 'hydroxyl function released on the adjacent phosphodiester bond. A large number of strategies for protecting the hydroxyls in 2 ′ have been developed and illustrate the difficulties in choosing the protection scheme for the hydroxyl groups.

(C. B. REESE. Nucleosides Nucleotides. 1985, 4, 117-127; R. KIERZECK. M. H.

CARUTHERS. C. E. LONGFELLOW, D. SWTNTON. D. H. TURNIER and S. M. FREIER.

Biochemistry. 1986. 25, 7840-7846; S. IWAI and E. OHTSUKA, Nucleic Acids Res., 1988.

16, 9443-9456:

C. LEHMANN, Y. Z. XU, C. CHRISTODOULOU, Z. K. TAN and M. J. GAIT. Nucleic

Acids Res., 1989, 17, 2379-2390; T. TANAKA, S. TAMATSUKURI. and M. IKEHARA.

Nucleic Acids Res., 1986, 14, 6265-6279.).

 The use of alkylsilyl protective groups and in particular tert-butyldimethylsilyl

(TBDMS) for 2 'hydroxyl facilitated the synthesis of ohgonbonucleotides. The TBDMS group is sufficiently stable under acidic or basic conditions and is easily removed by tetrα-butyl ammonium fluoride in THF to be used in the solid phase strategy (N. USMAN, KK OGILNIE, M: Υ. JIAΝG and RL

LEDERGEΝ. J. Am. Chem. Soc., 1987, 109, 7845-7854; Ν. USMAΝ. K. ΝICOGHOSIAΝ.

R. L. CEDERGREΝ and K. K. OGILNIE, Proc. Natl. Sa. USA, 1988, 85, 5764-5768 .: S. A.

SCARIΝGE, C. F. FRAΝCKLYΝ and Ν. USMAΝ, Nucleic Acids Res., 1990. 18, 5433-5441.X K. OGILNIE and M. J. ΝEMER, Tétrahedron Lett., 1980, 21, 4159: R T. POΝ and

K. K. OGILNIE, Nucleosides Nucleotides, 1984, 3, 485: R. T. POΝ and K. K. OGILNIE.

Tétrahedron Lett., 1984, 25, 713.).

The protection scheme used for oxygenated nucleosides was therefore applied in series ß oligothioribonucleon (Figure 8).

Namely the protections:

 - Benzoyl for adenine and cytosine. isoButyl for guanine as protection of exocyclic amino groups.

 - Dimethoxytrityl (DmTr) to protect the primary 5 'hydroxyls.

 - tert-butyldimethymsilyl (TBDMS) to protect the secondary hydroxyl in 2 '

- Methoxy for the protection of phosphates.

The first step makes it possible to protect the free hydroxyl in 5 ′ with a labile acid group, DmTr (Diagram 8). Thus the nucleoside derivative 22 is treated with dimethoxytrityl chloride (1.27 molar equivalents) in anhydrous pyridine under an inert atmosphere. After usual treatment of the reaction mixture, the dimethoxytrityl derivative 23 is purified by chromatography on silica gel and isolated with yields varying from 70 to 80%. The physicochemical characteristics of these derivatives 23 (NMR, Mass) are in agreement with the proposed structures.

m

 The abbreviations have the following meanings:

 a- B = T = Thymine

 b- B = U = Uracil.

 c- B = CBz = N-Benzoyl-4-Cytosine.

 d- B = ABz = N-Benzoyl-6-Adenine.

 e- B = GPal = N-Palmitoyl-2-Guanme,

Dmtr = Dimethoxy-4,4'-trityl. iPr = Isopropyl. Bz = Benzoyl. TBDMS = tert-butyldimethylsilyl. (iPr) ₂ P (Cl) OMe = Chloro. (N, N diisopropylamino, methoxyphosphine)

DIAGRAM 8

The second step (Diagram 8) consists in the protection of the secondary 2'-T-ydroxyl during gold silylation a mixture of 2'-O-silyl 24 and its regioisomer 3'-O-silyl 25 is obtained (KK OGILNIE and DW EΝTWISTLE, Carbohyd Res., 1981. 89, 203-2 10. KK OGILVIE, AL SCHIFMAΝ and CL PEΝΝEY, Can. J. Chem., 1979. 57, 2230 GH HAKIMELAHL ZA PROBA and KK OGILNIE, Can. J. Chem., 1982. 60, 1 106) These isomers must be separated with great care on a column of silica gel in order to to obtain the 2'-O-TBDMS 24 derivative with a purity greater than 99.95% measured by HPLC.

 Indeed if this is not the case. the subsequent stages will lead us to mixtures of oligothionucleotide 5'-3 'expected with the undesirable isomer 5'-2'.

Consequently, the purity of 5'-O-DmTr-2'-O-silyl 24 is essential for good synthesis.

Thus, obtaining the compounds 5'-DmTr-2'-O-TBDMS 24 was firstly carried out according to the -DOGILVIE and Coll method. (KK OGILVIE. AL SCHIFMAN and CL PENNEY. Can. J. Chem., 1979, 57, 2230.) which consists in condensing 23 with tert-butyldimethylsilyl chloride (TBDMSCI) (1.2 molar equivalent) in pyridine in the presence of 'imidazole (2.6 molar equivalents). At the end of the reaction, a mixture of 2'-O-TBDMS 24 derivative and of its 3'-O-TBDMS 25 isomer is obtained. The expected compound 24 is separated from the mixture by chromatography on a column of silica gel.

 It is then possible by an equilibration reaction to obtain 24 from its 3'-O-TBDMS isomer 25 (SS JONES and CB REESE. J. Chem. Soc Perkin Trans I. 1979. 2762.) This migration of the TBDMS grouping is intramolecular and takes place via an intermediate having a pentavalent silicon atom. The isomerization of the 3'-O-TBDMS compound obtained previously after a first chromatographic separation is carried out in ethanol at room temperature overnight. The two position isomers thus obtained are again separated by chromatography on a column of silica gel. This isomerization operation followed by purification is repeated three times.

 However, the yields of 5'-DmTr-2'-O-TBDMS 24 obtained by this method (40 to 50% depending on the bases) did not seem satisfactory to us. We used another, more recent, developed by OGILVIE (GH HAKIMELAHL ZA PROBA and KK OGILVIE, Can. J. Chem., 1982. 60, 1106.) which allows the preferential introduction of TBDMS in position 2 ' ribonucleosides ß This technique recommends the use of areent salts.

Thus 5'-dimethoxytrityl 23 is condensed with TBDMS chloride (1.3 molar equivalents) enηntsence of silver nitrate (1.2 molar equivalents) and 3.7 equivalents of pyridine in anhydrous THF. After purification by chromatography on a silica gel column, 56% yield is obtained in 24 and 27% in 5'-DmTr-3'-O-TBDMS 25

 Consequently, 5'-DmTr-2'-O-TBDMS 24 is obtained under these new conditions in a single step with a better yield, but the selectivity for introducing the TBDMS group at 2 'is not improved.

II.1.2 Synthesis of phosphoramidite derivatives 26 ad (Diagram 9) The four N-protected phosphoramidite β thioribonucleosides 26 ad are then obtained from the corresponding 5'-DmTr-2'-O-TBDMS β corresponding N-protected thioribonucleosides 24 (Scheme 9).

 The nucleoside derivative 24 was treated with chloro N, N diisopropylamino methoxy phosphine (2.5 molar equivalents) in the presence of N, N, N diisopropyl ethylamine (DIEA) in dichloromethane under an inert atmosphere. The phosphoramidite derivative 26 was purified by chromatography on silica gel and lyophilized in benzene, the phosphoramidites 26 a-d are obtained with yields varying from 78 to 80% depending on the nature of the base.

 DIAGRAM 9

Analysis of the ^{3 1} P and ¹ H NMR spectra of 26 clearly show that we obtain a single pair of diastereoisomers, which establishes the regioisomeric purity of the compounds obtained.

Indeed some authors (R. KIERZEK. MH CARUTHERS, CE LONGFELLOW. D. SWINION, DH TURNER and SM FREIER, Biochemistry, 1986. 25, 7840-7846.) Had hypothesized that the silylated groups in position 2 'n 'were not stable under the conditions of the phosphoiylaoon. However, U has been shown (WK KOHLER- W. SCHLOSSERM, G. CHARUBALA and W. PFLEIDLERER, Chemisiry and Biology υf Nucleosides and Nucleotides. Pfess Academy. New York. 1978, pp 347-358; K K. OGILVIE and DW ENTWISTLE. Carbohydrate Res. 1981. 89. 203-210: N USMAN. KK OGILVIE. MY JIANG and RJ CEDERGREN. J. Am. Chem. Soc, 1987, 109, 7845- 7854.) that the alkylsilyl groups migrate in protic solvents such as medianol or in aqueous solutions such as the pyridine / water mixture. but these silylated groups are stable in anhydrous solvents and in particular in non-pronounced bases such as anhydrous pyridine (KK OGILVIE. DW ENTWISTLE. Carbohydrate Res., 1981, 89, 203-210; W. KOHLER. W. SCHLOSSER. G. CHARUBALA and W. PFLEIDERER. Chemistry and Biology of Nucleosides and Nucleotides, Académie Press. New York, 1978, pp 347-358.). On the other hand, it has been clearly demonstrated in the ribonucleotide series that the use of 2'-O-silyl nbonucleosides in the synthesis of oligoribonucleotides leads exclusively to 5'-3 'bonds (KK OGILVTE. MJ NEMER. Tetrahedron Lett. , 1980, 21, 4159: RT PON and KK OGILVIE. Tétrahedron Lett., 1984, 25, 713; RT PON, and KK OGILVIE, Nucleosides Nucleotιdes, 1984. 3, 485. PJ GAREGG, I. LINDH, J. STAWTNSKI. and R. STROMBERG, Tétrahedron Lett., 1986, 27, 4055-4058). The synthesis of phosphoramidites is not stereospecific and the formation in equal quantities of the two diastereoisomers is due to the chirality of the phosphorus atom of configuration R or S. The formation of these two products is not troublesome insofar as subsequent oxidation reactions suppress this chirality.

II. 2. EXPERIMENTAL PART

General method for the preparation of 5'-O-dimethoxytrityl N-acyl 4'-thio-ß-D-ribonucleosides 23 a-d.

To a solution of N-acyl ß-D-thioribonucleoside 23 ad (1 mmol) in anhydrous pyridine (5.6 ml) is added dimethoxytrityl chloride (1.27 mmol, 430 mg). The reaction mixture is stirred at room temperature under an inert atmosphere for 90 to 120 min and then methanol (1 ml) is added. After 10 minutes of additional stirring, the reaction mixture is poured onto a saturated aqueous solution of sodium bicarbonate (25ml) and the products are extracted with dichloromethane (2 × 20ml). The organic phases are washed with water (2 × 100 ml) then dried over sodium sulfate and filtered. The filtrate is evaporated under reduced pressure and the residue obtained is chromatographed on a column of silica gel. The elution is carried out with a CH ₂ Cl ₂ / MeOH: 98/2 mixture in the presence of 1% triethylamine; the fractions containing the product are evaporated to dryness and the nucleosides 23 ad are obtained in the form of an oil. 1- [4'-thio-5'-O-dimethoιvtrityl-ß-D-ribofurannosyl] Thymine 23 a.

 Yield: 68%.

 Mass spectrometry FAB> 0 NOBA m / z = 577 [M + H] +, 303 [DmTr] +

¹ H NMR (DMSOdé, 360 MHz) δ 11.24, (s, 1 H, NH); 7.44, (s, 1 H, H ₆ ): 6.87-7.30. (m, 13 H, Aromatic H); 5.83, (d, 1H, H _{1 ',} J _{1', 2 '} = 7.0); 5.53, (d 1 H, OH _{2 '} , J _OH.2' = 5.8): 5.30, (d, 1 H, OH _{1 ',} J _{OH, 3'} = 4.7): 4.14, (dd, 1 H, H ₂ ', J _{2', 1 '} = 6.9. J _{2', 3 '} = 3.0): 4.00. (t 1 H, H ₃ ', J _{3', 2 '} = 3.0, J _{3', 4 '} = 3.0); 3.69, (s, 6H, OMe); 3.30, (dd, 1H, H _{4 '} ). 3.13. (m, 2 H, H _{5 '} , H _{5 "} ); L60, (s, 3 H, CH ₃ ). 1- [4'-thio 5'-O-dimethoιytrityl ß-D-ribofurannosyl] Uracil 23 b.

Yield 70%. ¹ H NMR (DMSOd ₆ , 360 MHz) δ 11.31, (s. 1 H, NH); 7.70, (d, 1 H, H ₆ , J ₆ , ₅ = 8.0). 7.40-6 89. (m, 13 H, aromatic H of the group dmTr); 5.84. (d, 1 H, H _{1 ',} J _{1', 2 '} = 5.7). 5.53. (d, 1 H, OH _{2 '} , J _{OH2', 2 '} = 5.6), 5.48, (d, 1 H, H ₅ , J _5.6 = 8.0); 5.26. (d, 1H, OH _{3 '} , J _{OH3', 3 '} = 4.45); 4.02. (m, 2 H, H _{2 '} , H _3' ): 3.74, (s. 6 H, OMe); 3.31, (m, 3 H, H ₄ ', H ₅ ', H ₅ ").

Mass spectrometry FAB> 0 NOBA m z = 563 [M + H] +, 303 [DmTr] - 1- [4'-thio 5'-O-dimethoxytrityl ß-D-ribofurannosyl] N-4-benxoylCytosine 23 c

 Yield 69%.

¹ H NMR (DMSOd _6. 250 MHz) δ 1 1.32. (s. 1H, NH); 8.40, (d, 1H, H ₆ , J _6.5 = 7.5); 8 01. (d, 2H, Ho of the benzoyl group): 7.63, (d, 1H, H ₅ , J _5.6 = 7.3); 7.44. (m, 17H, aromatic H from the DmTr group and Hm, p from the benzoyl group); 5.88. (D, 1H, H _{1 '} . J _{1', 2 '} = 4.1); 5.73, (d, 1H, OH _{2 ',} J _OH.2' = 5.2); 5.27, (d, 1H, OH _{3 ',} J _OH.H3' = 5.7), 4.06, (m, 1H, H _{2 '} , J _{2', 1 '} = 4.0, J _2' , _{3 '} = 3.7, J _2'.OH = 5.2); 4.03, (m, 1H, H _{3 ',} J _{3', 2 '} = 3.7. J _{3', 4 '} = 5.3. J _{3', OH} = 5.7); 3.76 (s. 6H, OCH ₃ of the DmTr group); 3.43, (m, 3H, H _{4 ',} H _5' and H ₅ "). Mass spectrometry FAB> 0 NOBA m / z = 666 [M + H] +, 517 [M + H-∅CONH ₂ ] + , 303 [DmTr] + 1- (4'-thio S'-O-dimethoxytrityl ß-D-ribofurannosyl] N6-benzoylAdenine 23 d

 Yield 70%

¹ H NMR (DMSOdé, 250 MHz) δ 1 1.25, (s. 1H, NH); 8.63, (s, 1H, H ₈ ); 8.57, (s. 1H, H ₂ ); 8.04. (d, 2H, Ho of the benzoyl group); 7.51, (m, 13H, aromatic H of the group DmTr): 6.92. (d, 3H, Hm, p of the benzoyl group); 5.98. (d, 1H, H _{1 '} , J _{1', 2 '} = 5.8): 5.75. (d, 1H, OH _{2 '} . J _{OH, 2'} = 3.5); 5.45. (d, 1H, OH _{3 '} , J _{OH, 3'} = 2.5); 4.70, (m, 1H, H _{2 '} ). 4.26. (m, 1H, H _{3 '} ); 3.74, (s, 6H, OCH ₃ of the DmTr group); 3.53, (m, 3H, H _{4 '} , H ₅ ' and H ₅ ")

Mass spectrometry FAB> 0 NOBA m / z = 690 [M + H] +, 303 [DmTr] +

General method for the preparation of 2'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl-N-acyl-ß-D-ribonucleosides 24 a-e and their 3'-TBDMS isomer 25 a-e.

To a solution of 5'-O-dimethoxytrityl 4'-thio N-acyl ß-D-ribonucleoside 23 ad (1mmol) in anhydrous THF (10ml) is added silver nitrate (1.2mmol. 203mg). The reaction mixture is then stirred for 5 minutes at room temperature before the addition of tert-butyldimethylsilyl chloride (TBDMSCI. 1.3mmole, 195mg) and anhydrous pyridine (3.7mmole, 0.193ml). Stirring at room temperature is then prolonged for 24 h. then the heterogeneous solution is filtered before being poured into an aqueous soludon of 5% sodium bicarbonate (30 ml). The products are extracted with dichloroma-hane (3x20ml) and the organic phases are washed with water then dried over sodium sulfate and filtered. The filtrate is evaporated to dryness and the residue is chromatographed on a column of silica gel using a mixture of CH ₂ Cl ₂ / AcOEt: 95 '5 as eluent, then of CH ₂ Ch / AcOEt: 80/20. The fractions containing the desired product (2'-O-TBDMS. 24 ad) are combined and evaporated to dryness. The 3'-O-TBDMS 25 ad isomer is then obtained.

1 - [2'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl-ß-D-ribofurannosyl) Thymine 24 a.

 Yield: 33%.

 Mass spectrometry FAB> 0 NOBA m / z = 691 [M + H] +, 713 [M + Na] +.

¹ H NMR (DMSOd ₆ , 300 MHz): δ 11.36, (s, 1 H, NH); 7.58, (s, 1 H, H ₆ ): 7.33-6.90. (m,

1:00 p.m., Aromatics): 5.89. (s. 1 H, H ₁ ' _, J _{1', 2 '} = 6.1); 5.27, (m, 1H, OH _{3 '} ): 4.20. (dd, 1

H, H _{2 ',} J _{2', 1 '} = 6.10, J _{2', 3 '} = 3.5): 3.97, (m, 1 H, H _3' ); 3.73, (s, 6 H, OMe): 3.36. (m, 1 H, H ₄ '): 3.25. (m, 2 H, H _{5 '} , H ₅ "); 1.58. (s. 3 H, CH ₃ ); 0.81, (s, 9 H, tert-butyl): 0.0. (d, 6 H,

Me ₂ If). 1- [2'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl-ß-D-ribofurannosyl] Uracil 24 b.

 Yield 56%.

Mp = 114 ° C.

¹ H NMR (DMSOdβ, 360 MHz): δ 11.20, (s. 1 H, NH); 7.81. (d, 1 H. H _6. J _6.5 = 8.1); 7.30-6.89. (m, 13 H, aromatic H of the group dmTr) 5.84, (d, 1 H, H _{1 '} , J _{1', 2 '} = 5.5): 5.50. (d, 1 H, H5. J _5.6 = 8.1): 5.23. (d, 1H, OH _{3 '} , J _{OH3', 3 '} = 4.7); 4.1 1, (q. 1 H, HT. J _{2 ', 1'} = 5.5. J _{2 ', 3'} = 3.5): 3.95. (m, 1H. H _{3 '} ): 3.74. (s. 6 H, OMe): 3.38. (m, 2 H, H _{4 '} , H _5' J _{4 ', 5'} = 3.28. (m, 1 H, H ₅ "): 0.82. (s. 9 H, tert-butyl); 0.05, (m , 6 H, Me ₂ Si).

 Mass spectrometry FAB> 0 NOBA m / z = 677 [M + H] +, 619 [M + H-teri-butane] -. 303 [dmTr] - 1- [2'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl-ß-D-ribofurannosyl] N4-benzoylCytosine 24 c.

 Yield 32%.

¹ H NMR (DMSOd ₆ , 250 MHz): δ 11.31. (s. 1H, NH); 8.51, (d, 1H, H ₆ , J _6.5 = 7.3): 8.01. (d, 2H, Ho of the benzoyl group): 7.51. (m, 14H, aromatic H of the group DmTr and H ₅ ): 6.93, (d, 3H, Hm, p of the benzoyl group): 5.93. (d 1H, H _{1 ',} J _{1', 2 '} = 3.0): 5.20. (D, 1H, OH _{3 '} , J _OH.H3' = 5.0); 4. 14. (m, 1H, H _{2 '} ): 4.00. (M, 1H, H ₃ '): 3.76. (S. 6H, OCH ₃ of the group DmTr); 3.38. (M, 3 H, H ₄ ', H _5' and H ₅ "): 0.86. (S. 9H, tert-butyl): 0 60. (d, 6H, Me ₂ Si).

Mass spectrometry FAB> 0 NOBA m / z 780 [M + H] ⁺ . 303 [DmTr] - 1- [2'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl-ß-D-ribofurannosyl] N6-benzoylAdenine 24 d.

Efficiency 35% ¹ H NMR (DMSOd ₆ , 250 MHz): δ 1 1.25, (s, 1H, NH); 8.64, (s, 1H, Hg); 8.61, (s. 1H, H ₂ ); 8.04. (d, 2H, Ho of the benzoyl group); 7.51, (m, 13H, aromatic H of the group DmTr): 6.95, (d, 3H, Hm, p of the group benzoyl); 6.01, (d, 1H, H _{1 ',} J _{1', 2 '} = 5.8); 5.48. (d, 1H, OH ₃ ', J _{OH, 3'} = 2.5): 4.70, (m, 1H, H _{2 '} ); 4.26, (m, 1H, H ₃ '); 3.75, (s. 6H, OCH ₃ of the group DmTr); 3.53, (m, 3H, H ₄ ', H ₅ ' and H ₅ "); 0.82, (s, 9H, tert-butyl): 0.50, (d, 6H, Me ₂ Si).

Mass spectrometry FAB> 0 NOBA m / z 804 [M + H] ⁺ , 303 [M + H] ⁺ 1- [3'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl ß- D-ribofurannosyl]

Thymine 25 a.

Yield 13%.

¹ H NMR (DMSOd ₆ , 360 MHz): δ 11.34, (s, 1H; NH); 7.46, (s, 1H, H ₆ ); 7.89-7.30. (m, 13H, aromatic H); 5.87, (d, 1H, H ₁ ', J _{1', 2 '} = 7.8): 5.46, (d, 1H, OH ₂ ', J _{OH, 2 '} = 5.1); 4 13. (m, 1H, H ₃ '): 4.11, (m, 1H, H _2' , J _{2 ', 1'} = 7.8, J _{OH, 2 '} = 5.1); 3.73, (s, 6H, OMe); 1.66. (s, 3H, CH ₃ ); 0.84, (s, 9H, tert-butyl); 0.50, (d, 6H, Me ₂ Si). 1- [3'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoxytrityl ß-D-ribofurannosyl]

 Uracil 25 b.

 Yield 27%

Mp = 109 ° C.

1 H NMR (DMSOdé, 360 MHz): δ 1 1.30, (s, 1 H, NH); 7.70, (d, 1H, H ₆ , J _6.5 = 8.0); 7.30-6.89. (13 H, aromatic H of the dmTr group): 5.84, (d, 1 H, H ₁ ', J _{1', 2 '} = 7.3): 5.57. (d, 1H, H5. J _5.6 = 8.0); 5.46, (d, 1 R OH _{2 '} , J _{OH2', 2 '} = 5.1): 4.12. (t 1 H, H _{3 '} J _{3', 2 '} = 3.1. J _3' , _{4 '} = 3.1); 4.03. (m, 1H, H _{2 '} ); 3.74, (s, 6H, OMe); 3.41, (m, 1 H, H ₄ ') - 3.21. (m, 2 H, H ₅ ', H ₅ "); 0.08, (s, 9 H, tert-butyl); 0.02. (m, 6 R Me ₂ Si).

 Mass spectrometry FAB> 0 NOBA m / z 677 [M + H] +, 619 [M + H-tert-butane] -, 303 [dmTr] +.

 This compound was brought into contact with a 0.25% (v / v) edianol / triethylamine solution for 12 h at room temperature to give a mixture of 24 b and 25 b in equal proportion. 1- [3'-O-tert-butyidimethylsilyl-4'-thio-5'-O-dimethoxytrityl ß-D-ribofurannosyl] N4-benzoyl Cytosine 25 c.

 Yield 31%.

¹ H NMR (DMSOd6, 250 MHz), δ 1 1.33, (s. 1H, NH); 8.40. (d, 1H, H ₆ , J _6.5 = 7 4); 8 01. (d, 2H, Ho of the benzoyl group): 7.60, (m, 14H, aromatic H of the group DmTr and H ₅ ); 6.92, (d, 3H, Hm, p of the benzoyl group): 5.92, (d, 1H, H ₁ ', J _{1', 2 '} = 5.3). 5.60. (D, 1H, OH _{2 '} , J _{OH, 2'} = 5.0); 4.15, (m, 2H, H _{2 '} , H ₃ '); 3.75, (s, 6H, OCH ₃ of the DmTr group); 3.38, (m, 3H, H ₄ ', H ₅ ' and H ₅ "); 0.79, (s, 9H, tert-butyl); 0.00, (d, 6H, Me ₂ Si) Mass spectrometry FAB> 0 NOBA m / z 780 [M + H] ⁺ , 303 [DmTr] ⁺ 1- [3'-O-tert-butyldimethylsilyl-4'-thio-5'-O-dimethoιytrityl ß-D-ribofurannosyl] N6-benzoyl Adenine 25 d.

 Yield 29%.

¹ H NMR (DMSOd ₆ , 250 MHz): δ 1 1.25, (s. 1H, NH); 8.64, (s, 1H, Hg); 8.61, (s, 1H, H ₂ );

8.04. (d, 2R Ho of the benzoyl group). 7.51. (m, 13H, aromatic H of the group

DmTr): 6.95. (d, 3H, Hritp from the benzoyl group); 6.01, (d, 1H, H ₁ 'J _{1', 2 '} = 5.8): 5.79.

(d, 1H, OH _{2 '} . J _{OH, 2'} = 3.5); 4.70. (m, 1H, HT); 4.26. (m, 1H, H _{3 '} ); 3.75, (s. 6R OCH ₃ of the group DmTr); 3.53. (m, 3H, H ₄ ', H ₅ ' and H ₅ "): 0.82, (s, 9H, tert-butyl): 0.01. (d, 6H,

Me ₂ If).

Mass spectrometry FAB> 0 NOBA m / z 804 [M + H] ⁺ , 303 [M + H] ⁺

General method for the preparation of β-D-ribonucleoside phosphoramidites 26 a-d.

To a solution of the compounds 24 a-d (1 mmol) in anhydrous dichloromethane (3.8 ml) are added under a led atmosphere of argon N, N, N-diisopropylethylaria (4 mmol. 0.7 ml). chloro N, N-diisopropylaminomethoxyphosphine (2.5mmole). 0.48ml) and 4-N, N-dimemylaminopyridine (0.2mmole, 24.4mg). The reaction mixture is stirred for 40 to 90 minutes at room temperature and is then diluted with ethyl acetate (35 ml). The resulting solution is washed with brine (4 × 50ml) and then with water (2 × 50ml). The organic phase is dried over sodium sulfate, filtered and evaporated under reduced pressure. The residue is chromatographed on a silica gel column and the elution is carried out using a mixture of cyclohexane, dichloromethane and methylamine (100/0 / 0.1 to 50/50 / 0.1). Products 26 a-d are obtained in the form of a white powder after lyophilization in benzene. 1- [2'-O-tert-butyldimethylsilyl-3'-N, N-diisopropyimethoxyphosphoramidite-4'thio-5'-O-diméthoιytrityl-ß-D-ribofurannosyl] Thymine 26 a (diastereoisomeric mixture) Yield: 89%.

³¹ p NMR (CD ₃ CN): δ 151.32 and 150.03.

¹ H NMR: δ 7.99, (s, 1 H, NH), 7.65, (d, 1 H, H ₆ ); 7.35-6.80, (m, 13 H, aromatic H): 5.97. (m, 1 RH _{1 '} ); 4.25, (m, 1 H, H _{2 '} ): 4.13, (m, 1 H, H ₃ '); 3.78. (s, 6 H, OMe): 3.68. (m, 1 H, H ₄ O: 3.54. (m 2 H, H ₅ ', H ₅ "); 3.35 (m, 3 H, MeO of the phosphoramidite group): 3.25, (m, 2 R CH of isopropyl groups ); 1.60, (m, 3 H, CH ₃ ): 1.15. (M, 12 H, CH ₃ of isopropyl groups); 0.86, (s. 9 H, tert-butyl); 0.05, (m, 6 H, Me ₂ Si). Mass spectrometry FAB> 0 NOBA m / z = 852 [MH] ⁺ 1- [2'-O-tert-butyldimethylsilyl-3'-N, N-diisopropylmethoxyphosphoroamidite-4'thio-5'-O -dimethoxytrityl-ß-D-ribofurannosyl] Uracil 26 b (diastereoisomeric mixture) Yield 78%.

NMR ^{3 l} P (CDCI ₃ ): δ 150.65 and 150.50 1 H NMR (CD ₃ CN, 250 MHz): δ 7.99, (s, 1 H, NH). 7.22, (m, 1 H, H ₆ ): 7.30-6.89. (m, 13 H, aromatic H of the group DmTr); 5.90. (m, 1H, H _{1 '} ); 5.50. (m, 1 H, H5): 4.13. (m,

2 H, H ₂ '. H3O; 3.80, (s, 6H, OMe from the DmTr group); 3.58. (m, 3 H, H ₄ ', H ₅ ', H ₅ "); 3.42.

(m, 2.H, 2 CH of isopropyl groups): 3.30 (m, 3H, MeO of the group DmTr).

1.19. (m, 12 H, CH ₃ of isopropyl groups); 0.84, (s, 9H, tert-butyl): 0.05. (m, 6 H,

Me ₂ If).

Mass spectrometry FAB> 0 NOBA m / z = 534 [M + H-DmTrH] ⁺ 1- [2'-O-tert-butyldimethylsilyl-3 ^, -N, N-diisopropylmethoxyphosphoroamidite-4'thio-5 -O-dimethoxytrityl -ß-D-ribofurannosyl] N4-benzoyl Cytosine 26 c

Yield 75%.

31 p NMR (CD ₃ CN): δ 151.30 and 150.05

Mass spectrometry FAB> O PEG m / z 941 [M + H] ⁺ . 637 [M + H-DmTrH], 303 [DmTr] ⁺ 1- [2'-O-tert-butyldimethylsilyl-3'-N, N-diisopropylmethoxyphosphoroamidite-4'thio-5'- O-dimethoxytrityl-ß-D- ribofurannosyl] N6-benzoyl Adenine 26 d

 Yield 71%.

NMR ^{3 1} p (CDCI ₃ ): δ 151.26 and 150.01

Mass spectrometry FAB> O PEG m / z 964 [M + H] ⁺ . 600 [M + H-DmTrH] 303 [DmTr] ⁺

II. 3 Functionalization of the solid support (Diagram 10).

The solid support or LCA-CPG (Long Chain AUcylamme Controlled Pore Glass) P-1 was first activated by a solution of 3% trichloroacetic acid in dichloromethane at room temperature for 2 to 3 hours in order to release the greater number of amino groups. which leads to the maximum of reactive sites on the surface of the glass beads. The support thus activated P-2 is then functionalized by coupling with succinic anhydride in pyridine in the presence of 4-DMAP which leads to P-3. In the next step, the 3'-silylated nucleoside 25 is used since it was obtained during the thioribonucleoside silylation reaction and is not necessary for the synthesis of oligothionucleotides containing the 3'-5 'phosphodiester bond, So. P-3 activated by 1- (3-dimethylaminopropyl) 3-ethylcarbodiimide (DEC) in the presence of 4-DMAP is reacted in anhydrous pyridine with 5'-O-DmTr-3'-OTBDMS ß-D-ttuoribonucleoside 25. Unused support sites are masked with piperidine. A final step of masking with acetic anhydride in the presence of collidine in THF leads to P-6. The quantity of nucleoside fixed on the support is determined by U spectrophotometry by measuring the quantity of dimethoxytrityl cation released after treatment with a 10% solution of trichloroaceric acid in dichloromethane. The degree of functionalization varies from 21 to 38 μmol per gram of support depending on the nature of the nucleoside base.

(i = TCA3%, CH ₂ CI ₂ ; ii = succinic anhydride, DMAP, Pyr .; iii = 5'-o-DmTr-4'-thio-3'-o- TBDMS-ß-D-ribonucleoside, DMAP, NEt ₃ , DEC Pyr .; iv = pentachlorophenol.

v = piperidine; vi = Ac ₂ O 0.5M in THF)

DIAGRAM 10 II. 4. Automated synthesis of thiooligonucleotides (Diagram 11).

 The thiooligonucleotides were synthesized on a DNA synthesizer (Applied Biosvstems model 381 A). The elongation cycle has four important stages.

 Detritylation of the nucleoside or oligonucleotide attached to the solid support.

 Condensation of phosphoramidite activated by tetrazole on the free 5 'hydroxyl of the nucleoside or oligonucleotide.

 Masking of unreacted 5'-hydroxyl functions.

Oxidation of phosphitetnester functions into phosphotriester.

DIAGRAM 11 - DEPROTECTION AND LOCKOUT

Each synthesis was carried out on a column containing 40 mg of solid support. The yield of each incorporation is 98% evaluated by measuring the concentration of the ethoxytrityl cations recovered after each coupling by treatment of the support with trichloroacetic acid.

Washings are carried out between each step, and the duration of an elongation cycle is 21.1 minutes for the synthesis of the homododecamer 4'-thio-ß-D-ribonucleotide (ßrSU ₁₂ ) (Table 1).

II. 5. Stall, Deprotection and Purification of the Thiooligomer

After having carried out the deprotection steps of the methoxyphosphate diesters with thiophenol. the detachment of the dodecamer from the solid support by ammonia, the deprotection of hydroxyls by TBAF. the thiooligomer was desalted on a G-25 DEAE Sephadex exclusion column or on an A-25 Sephadex DEAE ion column and purified by reverse phase HPLC.

II.6. EXPERIMENTAL PART. Example I: Synthesis of the homododecamer ßrSU ₁₂ .

I- 1 Synthesis of the solid support:

The solid support P-1 (2.415 g) is suspended with a solution (50 ml) of 3% trichloroacetic acid in anhydrous CH2Cl2. After 4:15 of agitation. 50 mJ of a triethylamine and diisopropylamine solution (9: 1) is added and after 5 minutes of stirring the solution is filtered. The solid support P-2 thus obtained is washed with dichloromethane (2 × 50 ml) then with ether (50 ml) and allowed to dry.

 The solid support P-2 is then suspended in anhydrous pyridine (14.5 ml) in the presence of succinic anhydride (0.48 g) and DMAP (0.0805 g). The suspension is stirred for 20 hours and then filtered. The solid support P-3 is then washed with anhydrous pyridine (50 ml), dichloromethane (2 × 50 ml) and to edify it (2 × 50 ml) then put to dry under vacuum.

Condensation of the solid support P-3 with 1- [3'-O-tert-butyldimethylsilyl-2'-hydroxy-4'thio-5'-O-dimethoxytrityl-ß-D-ribofurιnnosyl] Uracil 25 b

A mixture of P-3 (2,415 g) of 1- [3'-O-tert-butyldimethylsilyl-2'-hydroxy-4 'thio-5'-O-dimethoxytrityl-ß-D-ribofuraimosyl] Uracil 25 b (333 mg. 1 eq.) of DMAP (29.5 mmg. 0.5 eq.) of triethylamine (0.146 mg. 202 μl. 0.003 eq.) and DEC (920 mg, 10 eq) in 29 ml of anhydrous pyridine is stirred 3 days at room temperature.

 Pentachlorophenol (321 mg, 2.5 eq.) Is then added and left for a further 24 hours. The solid support is then filtered, rinsed with anhydrous pyridine (2 × 50 ml) then with dichloromethane (2 × 50 ml) and ether (2 × 50 ml)

Immediately after, the solid support P-5 is treated with 25 ml of pipendine After stirring at room temperature for 24 hours, the solid support is filtered. washed with dvichloromethane (2 × 50 ml) and then edited (2 × 50 ml) and dried under vacuum The dry solid support (2.415 g) is placed in the presence of a solution of acetic anhydride (0.5 M) in THF (15 ml) and of a solution of collidine (0.5 M) in THF (15 ml ). After 4 hours of reaction, the solid support is washed with anhydrous pyridine (2 x 50 ml), dichloromethane (2 × 50 ml), THF (2 × 50 ml) and ether (2 × 50 ml ),

P-6 thus obtained is dried under vacuum.

I-2) Determination of the functionalization rate of the solid support P-6.

This determination is carried out by assaying the DmTr groups released in an acid medium. We weigh exactly 38.0 mg of P-6 which are suspended in 3.4 ml of a solution of para-toluene sulfonic acid (0.1 M) in acetonitrile. . The mixture is stirred for 15 min and then sonicated for 2 min.

 The volume is then adjusted to 10 ml with the 0.1 M solution of para-toluene sulfonic acid in acetonitrile. 0.3 ml of this solution is taken, to which 5 ml of the 0.1 M solution of para-toluene sulfonic acid are added. Reading the OD (0.317 OD unit) at 500 nm allows us to calculate the degree of functionalization of the P-6 support, which in this case is 21 μmol / gram of solid support.

I-3) Automated union of the homododecamere ßrSU ₁₂

The synthesis is carried out on a column containing 39.8 mg of solid support functioning at 21 μmol / g of resin, ie 0.835 μmol (1 eq) of 4'-thioundine.

 Each elongation cycle requires an excess of phosphoramidite synthon (20 μmole. 24 eq), ie 240 μmole in total for the synthesis of the dodecamer. The synthesizer taking 200 μl of a 0.1 M solution of synthon in acetonitrile by incorporation.

 The duration of an elongation cycle is 21.1 min (Table 1).

 The coupling step has been considerably increased (900 s versus 45 s in the case of a deoxynucleoside) due to the lower reactivity of the synthon nbonucleotide

 The assay of the dimethoxytrityl cations released at each detritylation stage made it possible to evaluate the average efficiency of incorporation of the thioribonucleonic unit at 98.7%.

I-4) Detachment, deprotection and purification of the homododecamer duo ßrSU ₁₂

The solid support carrying the thio-homododecamer ßrSU ₁₂ is treated with 5 ml of a thiophenol solution. triethylamine. dioxane 1. 2 (1/2/2) for half an hour at room temperature.

 The thiophenol solution is then filtered and the support washed with an ammonia solution

32% in edianol 95 (3/1: v / v) (3 × 500μl) to unhook the thio-homododecamere from the solid support. The solution thus obtained is evaporated, taken up in 500 μl of water and then lyophilized. The oligomer is then dissolved in 300 μl of a solution of TBAF (1.1 M) in THF. The reaction mixture is left under stirring for 24 hours. The reaction is then stopped with 300 μl of an aqueous solution of ammonium acetate (0.05 M).

The solution is evaporated to dryness. coevaporated 3 times with water and the residue chromatography on a column of Sephadex G-25 exclusion gel. The fractions containing the thioohgomere are combined, evaporated to dryness and analyzed by HPLC,

Analysis and Purification of the Thio-Oligomer ßrSU ₁₂ Obtained.

The HPLC analysis of the thiooligomer was carried out on a Beckman C-18 RP 3 μ x LODS Ultrasound column under the following gradient conditions:

 A. 10% acetonitrile in 0.05 M aqueous solution of triethylammonium acetate

B. 15% acetonitrile in a 0.05 M aqueous solution of triethylammonium acetate Gradient:

A- - - - - - - - - - - -B - - - - - - - - -B - - - - - - - - - A

 20 '10' 5 '

 Analysis time: 30 '.

Under these conditions, it is found that the thiooligomer is 92.73% pure.

A purification of ßrSUi2 is then carried out on a Nucleoss 1 semi-preparative column with a gradient:

 A- - - - - - - - - - - - - -B - - - - - - - - - -B - - - - - - - - - - -A

 15 '10' 5 '

 Under these conditions, the ßrSUπ is obtained with a purity of 94.42% sufficient for subsequent studies.

Example II: Synthesis of the dodecamer ß (SrT) ₁ dT ₁₁ .

II-1) Synthesis of the solid support.

The solid support functionalized at 1 μmol for 35 mg of 2'-deoxythymidine resin as well as the synthons 1- [5'-O-DmTr-3'-O-N, N-diisopropylamino cyanoethyl phosphine. 2'-deoxy-ß-D-ribofurannosyl thymine are marketed by Applied Biosystems

II-2) Automated synthesis of ß (SrT) ₁ dT ₁₁ .

The symphesis is carried out on a column containing 35 mg of solid support, ie l μmol of 2'-deoxythymidine. Each elongation cycle requires an excess of phosphoramidite synthons (20 μmoles. 20 eq), ie 220 μmol of 2'-deoxythyriudine synthons and 20 μmoles of 4'-thio-thymidine synthon 5. The synthesizer taking 200 μmoles by incorporation of a 0.1 M solution of each synthon in acetonitrile. The duration of a cycle of incorporation of a deoxythymidine unit is 5.5 minutes. While it is 20.1 minutes for a cycle of incorporation of the 4'-thiothymidine syntlion.

 The increase in the duration of the cycle of incorporation of the chimeric syntiion is due to the coupling step 909 'against 36' for the synthon 2'-deoxythymidine.

The assay of the dimethoxytrityl cations released at each detritylation stage made it possible to evaluate the average yield of incorporation of the 2'-deoxythymidine unit at 98.5% and 93.5% for the synthon 4'-thiothymidine.

II-3) Unhooking deprotection and purification of ß (SrT) ₁ dT ₁₁ The solid support carrying the ß (SrT) ₁ dT ₁₁ oligomer is treated with an ammonia solution (32%) in edianol 95 (3 / 1. v / v) (3 × 500 μl) for 3 cycles of 20 min each in order to unhook the oligomer from its support, then the dodecamer is incubated for 2 hr in a dry oven in the same mixture in order to deprotect the functions cyanoediyl and methoxy.

 The oligomer is then evaporated in vacuo, taken up in 500 μl of water and lyophilized

Deprotection of the TBDMS groups is effected according to the protocol developed in Example I.

The residue obtained is chromatographed on a DEAE Sephadex A-25 gel column. The fractions containing the oligomer are combined, evaporated to dryness and analyzed by CLPH Analysis and Purification of ß (SrT) ₁ dT _{1 1} by CLPH,

HPLC analysis of the oligomer was carried out on a Beckman C 18 RP 3 μ column

Ultrasound under the following gradient conditions:

 A: 6% acetonitrile in a 0.05 M methyl ammonium acetate buffer.

B: 20% acetonitrile in a 0.05 M triethyl ammonium acetate buffer.

A- - - - - - - - - - - - - - - - -B- - - - - - - - - - - - - - - B

 15 '10'

 Analysis time: 25 min.

Under these conditions, the oligomer is 49.3% pure.

a purification of ß (SrT) ₁ dT _{1 1} is then carried out by semi-preparative HPLC with a Nucleosyl column under the following gradient conditions:

 A- - - - - - - - - - - - - - - B- - - - - - - - - - - - - -B

 15 '5'

 The 12-mer then has a retention time of 12.99 min and a purity greater than 99%.

H, 4 .: Synthesis of the dodecamer ß to T ₅ (SrT) dT ₆ II-4.1) Synthesis of the solid support

The solid support defined in Example II was unhsé. II-4 2) Automated synthesis of ß dT ₅ (SrT) dT ₆ .

The same synthesis conditions developed for the synthesis of ß (SrT) ₁ dT ₁₁ have been used.

 The assay of the dimethoxytrityl cations released at each detritylation stage made it possible to evaluate the average yield of incorporation of the 2'-deoxythymidine unit at 99 3% and at 96.1% for the synthon 4'-thiothymidine.

II-4.3) Detachment and purification of ß dT ₅ (SrT) dT ₆

The treatment of this ohgomer was carried out according to the protocol developed in 1 example II Analysis and purification HPLC. carried out under the same gradient conditions reveals a retention time of 13.18 minutes and a spectrophotometric purity at 260mm of 100

III. AUTOMATED SYNTHESIS OF 4'-THIO-OLIGORIBONUCLEOTIDES AND

MIXED OLIGOMERS.

We describe the synthesis of a homogeneous 4'-dιio-ribo ohgomere 1 dinge on the splice sequence accepting the tat HIV gene, We show that it is then possible to obtain mixed oligomers comprising a limited sequence of DNA with phosphodiester (sequence 4) or phosphorodioate (sequence 5) linkages

 Such 5'- (4'-S-RNA / DNA / 4'-S-RNA) -3 '4 and 5 mixed sequences can be used in an antisense approach and are likely to be highly selective as far as "window" of DNA structure will be the only substrate of RNAse H after apartment of the antisense with a complementary target. It should be noted that the DNA "window" (phosphodiester or phosphorothioate) can be of variable length - in sequences 4 and 5 there are six deoxynucleotides - and that this "window" can be used at any position of the oligomer, even at the 5 'and / or 3' ends.

 In addition, this possibility opens the way to the design of amorphous enzymes.

(homogeneous or not) comprising 4'-S-RNA oligo-ribonucleon sequences

A number of homogeneous 4'-duo-ohgoribonucleons (4'-S-RNA) were syndiosed on a DNA synthesizer (Applied Biosystems model 381 A) using the general procedure described above.

This is particularly the case: of 4'-Sr- (ACACCCAAlUCU) 1

from 4'-Sr- (UUUUUU), (4'-SrU ₆ ), 2

and 4'-Sr- (UUUUUU-3'-P-CH ₂ -CH ₂ -CH ₂ -OH), (4'-SrU ₆ -3'n-prOH) 3

The expression 4'-Sr means: oligoribonucleotide of configuration β where the oxygen of the furanose cycle of sugars is replaced by a sulfur atom.

 3'n-prOH means that an n-propanol function is fixed on the phosphate group introduced at the 3 'end of the oligoribonucleoride.

In addition, mixed oligomers comprising sequences of phosphodiester or phosphorothioate oligodesoxynucleotides were obtained and the modifications made to the elongation cycle are described by taking the following sequences as an example.

4'-Sr- (UUU) -d (TTTTTT) -4 ^, -Sr- (UUU) / P _{1 1} ,

[4'-SrU ₃ -dT ₆ -4'-SrU ₃ / P _{1 1} ] 4

and 4'-Sr- (UUU) -d (TTTTTT) -4'-Sr- (UUU) / P ₃ PS ₅ P ₃ ,

[4'-SrU ₃ -dT ₆ -4'- SrU ₃ / P ₃ PS ₅ P ₃ ] 5

or 4'-Sr has already been defined as above, P _x corresponds to the number of phosphodiester bonds between the nucleotide units and PS _y to the number of phosphorothioate bonds between the nucleotide units.

 The thio-oligoribonucleotides were synthesized on a DNA symhetizer (Applied Biosystems model 381 A).

1II-1) Synthesis of -f'-ιhιoolιgorιbonucleotιdes: -f'-Sr-fA-CACCCAAUUCU) 1. 4'-SrU ₆ , 2, and 4 -SrU ₆ -3'n-prOH 3

The elongation cycle comprises four important stages: a) Detritylation of the nucleoside or of the oligonucleotide fixed on the solid support in the case of 4'-Sr116 and 4'-Sr- (ACACCCAAUUCU), or else detritylation of the arm n -propanol fixed on the solid support or of the oligonucleotide fixed on the n-propanol link in the case of 4'-SrU ₆ -3'n-prOH, b) Condensation of the phosphoramidite activated by tetrazole on the free 5 'hydroxyl nucleoside or oligonucleotide. in the case of 4'-SrU ₆ and 4'-Sr- (ACACC- CAAUUCU) or on the free hydroxyl of the n-propanol arm or on the hydroxyl in 5 'of the oligonucleotide in the case of 4'- SrU ₆ -3'n-prOH, c) Masking of unreacted 5'-hydroxyl functions. d) Oxidation of the phosphitetriester functions to phosphomester. Each synthesis was carried out on a column containing 40 mg of solid support. The yield of each incorporation is 98% evaluated by measuring the optical density of the dimethoxytrityl cations released after each coupling by treatment of the support with trichloroacetic acid.

 Washes are carried out between each step, and the duration of an elongation cycle is

21.46 minutes for the synthesis of the three aforementioned oligomers. (Table 2).

III-2 Synthesis of mixed oligomers: 4'-SrU ₃ -dT ₆ -4'-SrU ₃ / P ₁₁ 4 and 4'-SrU ₃ -dT ₆ -4'-SrU ₃ / P ₃ PS ₅ P ₃ .

III-2-1 Oligonucleotide 4 '-S _rU3 -dT ₆ -4' -SrU ₃ P ₁₁ 4.

The automated synthesis of this oligonucleotide repeats the four steps described in III. Each synthesis was carried out on a column containing 40 mg of solid support. The yield of each incorporation is 98%. The duration of an elongation cycle per 4'-Sr-U monomer is 21.43 minutes compared to 6.73 minutes for a dT monomer This difference is due to the increase in the coupling time of a 4'-Sr- enrite U which is 91 5 seconds against 36 seconds for a dT unit. (table 3).

III-2-2 Oligonucleotide 4 '-SrU ₃ -dT ₆ -4' -SrU ₃ P ₃ PS ₅ P ₃ 5.

The same conditions of automated synthesis were applied to this mixed dodecamer comprising both phosphodiester and phosphorothioate bonds.

 Syndiation was carried out on the scale of a μmole, ie on a column containing approximately 40 mg of solid support functionalized at 21 μmole / g. The average incorporation yield is 98%.

 During the introduction of a phosphorothioate type nucleotide bond. the oxidation step defined in paragraph IV-2-1 (step 4. Table 3) has been replaced by a 51 second sulfurization step using Beaucage reagent ("derivatives of benzene carboxythioanhydride sulfone") dissolved in acetonitrile has a concentration of 0.05 M.

 The other stages of the elongation cycle remain unchanged.

III-3 Detachment, Deprotection and Purification of 4-thiooligoribonucleotides (4'-S-RNA). After having carried out the steps of deprotection of the methoxyphosphotriesters into phosphodiesters by treatment with thiophenol, the 4'-thiooligoribonucleotides were separated from the solid supports by a treatment with ammonia. The TBDMS groups introduced on the hydroxyls in 2 ′ were deprotected by a solution of TBAF in THF. The thiooligomers are then desalted on a DEAE Sephadex G-25 exclusion column and then purified by reverse phase HPLC.

III-4 EXPERIMENTAL PART.

Example I: Synthesis of hexamers: 4'-SrU ₆ 2 and 4'-SrU ₆ -3'n-prOH, 3.

I-1 Synthesis of solid supports: The solid support necessary for the synthesis of 4'-SrU ₆ , 2 is the same as that used for the synthesis of ßrSU _{12 '} .

The synthesis of 4'-SrU ₆ -3'n-prOH 3 requires the use of the universal solid support

 From intermediate P-3 described in Patent No. 92,01275, a mixture of 10-DmTr-propanol-3 (1 eq.), DMAP (29.5 mg, 0.5 eq.), triethylamine (0.146 mg, 0.003 eq.) of DEC (920 mg, 10 eq.) and P-3 (2.415 g) is stirred in 29 ml of anhydrous pyridine for three days at room temperature. Pentachlorophenol (321 mg, 2.5 eq.) Is added and stirring of the reaction mixture is continued for an additional 24 hours. The solid support is then filtered, rinsed with anhydrous pyridine (2 × 50 ml) then with dichloromethane (2 × 50 ml) and ether (2 × 50 ml).

 The solid support is then treated with piperidine, then with a solution of acetic anhydride and collidine in THF according to the protocol defined above.

I-2) Determination of the functionalization rate of the solid support.

This determination was made using the procedure described above. The functionalization rate of the solid support was evaluated at 16.4 μmol of n-propanol per gram of resin. I-3-) Automated synthesis of hexamers 4'-SrU ₆ 2 and 4'-SrU ₆ -3'n-prOH 3.

I-3-1) Automated synthesis of the hexamer 4'-SrU ₆ 2. The synthesis is carried out on a column containing 39.4 mg of solid support functionalized with 21 μmol / g of resin, ie 0.827 μmol (1 eq) of 4 '-thiouridine. Each elongation cycle requires an excess of phosphoramidite synthon (16.75 μmole. 20.3 eq), ie 100.5 μmole in total in 0.1 M solution in acetomtrile. The duration of the elongation cycle defined in table 1 is 21.46 minutes.

The assay of the dimethoxytntyies cations released at each detritylation stage made it possible to evaluate the average yield of incorporation of a thioribonucleotide unit at 98.3%.

I-3-2) Automated synthesis of the hexamer 4'-SrU ₆ -3'n-prOH 3.

The synthesis is carried out on a column containing 36.2 mg of solid support functionalized with 16.4 μmole of n-propanol per gram of resin, ie 0.82 μmole (1 eq) of n-propanol.

The total synthesis of 4'-SrU ₆ -3'n-prOH, 3 requires 95.9 mg of phosphoramidite synthon

(1 14.8 μmol. 20 eq.) Dissolved in 1.14 ml of acetonitrile. The length of an elongation cycle is 21.46 minutes.

 The average yield per coupling is 98.3%.

I-4) Detachment, deprotection and purification of 4'SrU ₆ 2 and 4'-SrU ₆ -3'n-prOH 3.

The treatment successively using thiophenol of ammonia and a solution of TBAF. common to both hexamers is previously decnt.

I-4-1) Analysis and Purification of the hexamer 4'-SrU ₆ 2.

The HPLC analysis of the thiooligomer was carried out on an SFCC Nucleosy l C-18 N 125 2P 789 column under the following gradient conditions:

 A: 10% acetonitrile in 0.05 M aqueous solution of triethylammonium acetate B: 15% acetonitrile in 0.05 M aqueous solution of triethylammonium acetate

Gradient:

Analysis time: 30 minutes, flow rate: 1 ml / min. Under these conditions, the hexamer 4'-SrU ₆ , 2 has a spectrophotometric purity at 260 nm of 95.3%.

Purification by preparative HPLC on a semi-preparative nucleosyl coionne with a flow rate of 2 ml / min and an isocratic eluent of 12% acetonitrile in an aqueous 0.05 M triethylammonium acetate solution allows us to obtain the 4 '-SrU ₆ , 2 with a spectroscopic purity of 97.7%.

I-4-2) Analysis and purification of the hexamer 4'-SrU ₆ -3'n-prOH 3.

The analysis is carried out under the same column and gradient conditions as for the 4'-SrU ₆ hexamer.

4'-SrU ₆ -3'n-prOH, 3 was obtained with a spectroscopic purity at 260 nm of 51.6% This purity is brought to 92% after purification by preparative HPLC carried out in a gradient of 10% to 13% d acetonitrile in 0.05 M aqueous triethylammonium acetate solution in 15 minutes, followed by 13% acetonitrile in 0.05 M aqueous triethylammonium acetate solution for 10 minutes

Example II: Synthesis of the mixed dodecamer: 4'-SrU ₃ -dT ₆ -4'-SrU ₃ / P _{1 1} 4

II-1) Synthesis of the solid support. The solid support used for this symmetry is identical to that necessary for the synthesis of ßrS U ₁₂

II-2) Automated synthesis of 4'-SrU ₃ -dT ₆ -4'SrU ₃ / P ₁₁ 4.

The synthesis of the dodecamer is carried out on a column containing 39.3 mg of solid support, ie 0.819 μmol of 4'-thiouridine. Each elongation cycle requires an excess of 4'-thiouridine phosphoramidite synthons (16.75 μmoles. 20.3 eq) or 100.5 umole of 4'-thiouridine synthons dissolved in 1.2 ml of acetonitrile (0.083 M) and an excess of desoxythymidine phosphoramidite synthon ( 20 cq .. 16.38 μmole) or 98.28 μmole of dT in 0.1 M solution in acetonitrile. The duration of a cycle of incorporation of a deoxythymidine unit is 6.73 min. whereas it is 21.43 min for a cycle of incorporation of the synthon 4'-thioribouridine.

 The assay of the dimethoxytntyl cations released at each detntylation step made it possible to evaluate the average incorporation yield at 98.0%.

II-3) Detachment and purification of 4'-SrU ₃ -dT ₆ -4'SrU ₃ / P ₁₁ 4. The stages of detaching the oligomer from the solid support and deprotecting the internucleotide phosphotriester bonds with thiophenol are identical to those described in Example I. II-3-1) Analysis and Purification of 4 'SrU ₃ -dT ₆ -4 '-SrU ₃ P ₁₁ 4 by HPLC.

HPLC analysis of oligomer 4 was carried out on an SFCC Nucleosyl C 18 N 125 column

2P789 in a gradient of 10% to 15% of acetomtrile in an aqueous solution of 0.05 M triethylammonium acetate for 20 minutes (flow rate: 1 ml / min. Analysis time. 30 min) Under these conditions, the mixed dodecamer has a specnOphotometric purity at 260 nm of 92.5%.

Purification by semi-preparative HPLC on a Nucleosyl column with a flow rate of 2 ml / min in the same gradient increases the spectrophotometric purity of the mixed sequence 4'-SrU ₃ -dT ₆ -4'SrU ₃ / P ₁₁ 4 to 99 , 5%.

EXAMPLE III Automated synthesis of 4'-SrU ₃ -dT ₆ -4'-SrU ₃ / P ₃ PS ₅ P ₃ 5. III-1) Synthesis of the solid support.

The solid support used is the same as that described in Example I.

III-2) Automated synthesis of 4'-SrU ₃ -dT ₆ -4'SrU ₃ / P ₃ PS ₅ P ₃ 5.

The synthesis of the dodecamer is carried out on a column containing 39.5 mg of functionalized support, ie 0.831 μmol (1 eq) of 4'-thiouridine. Each elongation cycle requires an excess of synthon 4'-thioundine phosphoroamidite (16.75 μmole. 20.3 eq.) Or 100.5 μmole of 4 'Sr-U in total and an excess of 20 eq. (16.38 μmole) of deoxythymidine synthon, that is 98.28 μmole for the entire synthesis.

 The duration of a deoxythymidine incorporation cycle is 7.7 minutes compared to 21.43 minutes for the incorporation of a 4'-thioundine. The assay from dimethoxytntyl cations released indicates an average yield of synthons incorporation of 98.7%.

III-3) Unhooking, deprotection and purification of 4'-SrU ₃ -dT ₆ -4'-SrU3 / P ₃ PS ₅ P ₃ 5.

The treatment of this oligomer is identical to that defined in Example I. HPLC analysis and purification, carried out on an SFCC Nucleosyl C18 N 125 2P789 column in a gradient of 10% to 20% acetonitrile in an aqueous solution 0.05 M methylammonium acetate for 20 mmutes (flow rate: 1 ml / min. analysis time 30 min). shows a spectrophotometric purity at 260 nm of 85%. Purification by semi-preparative HPLC on a semi-preparative Nucleosyl column with a flow rate of 2 ml / min under isocraric conditions at 19% acetomtrile in an aqueous solution of 0.05 M triethylammonium acetate increases the purity of l 'oligomer 99]%.

Example IV: Synthesis of the anti-tat dodecamer:

4'-Sr- (A-C-A-C-C-C-A-A-L-U-C-U) 1.

IV- 1) Synthesis of the solid support.

The solid support used for this automated synthesis is identical to that described in Example 1.

IV-2) Automated synthesis of the anti "tat" dodecamer:

 4'-Sr- (A-C-A-C-C-C-A-A-U-U-C-U) 1.

The synthesis of this heterododecamer complementary to the V1H tat gene splice acceptor site is carried out on a column containing 38.5 mg of functionalized solid support sou on 0.808 μmol of 4'-thioundine (1 eq.). Each elongation cycle uses an excess of

 25.24 eq (20.4 μmol) in synthon 4'-thioadenosine phosphoroamidite. 13.5 eq (10.97 μmole) in synthon 4'-thiocytidine and 30 eq. (24.25 μmole) in 4'-thiouridine synmon

 The duration of an incorporation cycle is 20.71 minutes and the assay of the dimethoxytrityl cations indicates an average yield of incorporation of the phosphoramidite synthons of 93.6%.

IV-3) Unhooking, deprotection and purification of 4'-Sr- (A-C-A-C-C-C-A-A-U-L-C-L) 1.

The solid support carrying the dodecamer 4'-Sr- (ACACCCAA-L'-UCU), 1 is treated with 5 ml of a thiophenol / triethylamine / dioxane solution (1/2/1) for half an hour at room temperature . The thiophenol solution is then filtered and the solid support is washed with 3 × 3 ml of metbanol. then with a solution of 32% ammonia in ethanol 95

(3/1: v / v) (3 × 500 μl) in order to unhook the oligomer from its support. The solution obtained is incubated in a dry thermostat-controlled oven at 55 ° C for 5 hours in order to deprotect the protective groups from heterocyclic bases. It is then evaporated. taken up in 500 μl of water and lyophilized. The lyophilized oligomer is then dissolved in 300 μl of a solution of TBAF (1.1 M) in THF. The reaction mixture is left at room temperature for 24 hours. The reaction is then stopped with 300 μl of an aqueous 0.05 M ammonium acetate solution. The solution is evaporated to dryness. co-e vaporee with 3 portions of 500 μl of water and the residue is desalted on a column of exclusion gel DEAE Sephadex G-25. The fractions containing the 4'-thiooligomer are combined _and evaporated to dryness. redissolved in 1.2 ml of water and filtered through a millipore filter before being analyzed by HPLC, IV-4) Analysis and purification of 4 -Sr- (ACACCCAAUUCU) 1.

The analytical HPLC analysis of dodecamer 1 was carried out on a SFCC Nucleosyl C18 N125 2P789 column in a gradient of 10% to 15% of acetonitrile in an aqueous solution of 0.05 M triethylammomum acetate with a flow rate of 1 ml. / min and an analysis time of 30 minutes. The chromatogram indicates a spectrophotometric purity at 260 nm of 44.9%. Purification by semi-preparative HPLC on a semi-preparative Nucleosyl C18 column using the same gradient with a flow rate of 2 ml / min and an analysis time of 30 minutes brings the spectroscopic purity of 4'-Sr- (ACACCCAAUUCU) , 1 to 100%.

IV. PROPERTY OF HYBRIDIZATION OF β-4'-THIO-OLIGONUCLEOTIDES.

UV spectrophotometry makes it possible to study the nucleic acids and to highlight the formation of duplexes, the melting temperature being one of the characteristics of a duplex. UV absorption (Hypochromicity). Thus by spectrophotomeme U. V. it is possible to control the formation or the dissociation of a duplex. (W SAΝGER. Principles of Nucleic Acid Structure. Springer-Verlag, .Νew-York, 1984. pp 141-149) When the temperature of a solution containing a double helix (DNA or RNA) is slowly increased, UV absorption clearly believes throughout the dissociation. The inflection point of the sygmoidal curve of the variation in absorbance as a function of temperature is called melting temperature or Tm and corresponds to the coexistence of separate strands and strands paired in a 50/50 ratio. .

 Analysis of the fusion curves of the ß-4'-thiooligoribonucleonde / ß-oligodeoxyribonucleotide and ß-4'-thiooligoribonucleotide / ß-oligoribonucleonde duplexes will make it possible to define the thermal stability of such duplexes by measuring the melting temperature. These hybridization experiments are carried out while respecting a 1/1 stoichiometry between the two complementary strands, in the presence of a 1 M ΝaCl buffer. 10 mM sodium cacodylate, A high concentration of ΝaCl indeed favors the pairings in solvatarit the negative charges around the phosphorus atoms of the two oligonucleotide We therefore carried out these hybridization experiments in another buffer: 0.1 M NaCl. 10 mM sodium cacodylate, IV. 1. RESULTS AND DISCUSSION

Examples I and II: Fusion Experiments of the ßSrU ₁₂ / PolyrA Duplexes and ßrU ₁₂ / polyrA.

These experiments would highlight the influence of the sulfur atom in the 4 'position of the modified dodecamer on the thermal stability of the duplex by comparing the Tm obtained with that of the natural duplex

 Table 4 summarizes the melting temperatures obtained in the two experiments

Analysis of these results show that the βSrU ₁₂ / polyrA duplex has good thermal stability represented by the Tm of 46 ° C. obtained at a concentration of 1 M in NaCl

Examples III and IV: Fusion Experiments of ßSrU ₁₂ / ßdC ₂ At ₁₂ C ₂ and ßrU ₁₂ / ßdC ₂ At ₁₂ C ₂ Duplexes.

The melting temperatures obtained in the two experiments are summarized in Table 5.

Example IV: Self-hybridization experiment of the ßSrU ₁₂ oligomer

In order to verify the preceding results, it is necessary to ensure that the dodecamer ßSrU ₁₂ does not lead to any autohybridaoon. For this, a self-hybridization experiment of the dodecamer ßSrU ₁₂ was carried out at two NaCl concentrations (1 M and 0 1 M) The hybridization curves do not have the characteristic sigmoidal appearance but obey equation A = kT or A is the absorbance at 260 nm, T the temperature ci k a constant. No point of inflection can be visualized on such a straight line. We can therefore conclude that the dodecamer ßSrU ₁₂ does not self-pair. The comparison of the melting temperatures of the duplexes ßSrU ₁₂ / ßdC ₂ A ₁₂ C ₂ (Table 5) and ßSrU ₁₂ / polyrA (Table 4) shows a thermal stability of the RNA7ARN homoduplex (Tm = 46 ° C) significantly higher than that RNA / DNA heteroduplex (Tm = 27 ° c) on the other hand, no self-hybridization of ßSrU ₁₂ was observed.

Example VI and VII: Fusion Experiment for ßdT ₅ Duplex (SrT) dT ₆ / ßdC ₂ A ₁₂ C ₂ and ßdT ₁₂ / ßdC 2A ₁₂ C ₂

This experiment makes it possible to evaluate the thermal subsistence of the duplex ßdT ₅ (SrT) dT ₆

/ ßdC ₂ A ₁₂ C ₂ compared to that of the natural duplex ßdT ₁₂ / ßdC ₂ A ₁₂ C _2.

 (Figure 1). The melting temperatures of these duplexes are shown in Table 6.

 In view of these results, it appears that the thermal subsistence of the modified duplex is lower than that of the natural duplex. However, the difference of 5 ° C existing between the two melting temperatures is too small to conclude that there is a mismatch. Indeed, some authors (Y.KAWASE, S.IWAL H, INOUE, K.MIURA and E.OKTSUKA. Nucleic Acid Research, 1986, 14, 7727) have shown that the existence of a single mismatch in a duplex of 13 nucleotide units resulted in a decrease in Tm greater than 10 ° C.

We can therefore conclude that the 4'-thio-nucleotide unit pairs well with 2'-deoxyadenosine but with an affinity lower than that of 2'-deoxythymidine as confirmed by the difference in Tm of 5 ° C obtained ( Table 6).

IV. 2. EXPERIMENTAL PART

General method for hybridization experiments A - Determination of complementary oligomers In order to carry out the hybridization experiment. it is necessary to know the concentration of each oligomer to respect the 1: 1 stoichiometry between the two complementary strands. According to BEER-LAMBERT's law A = εlc. the absorbances of oligomers were measured at 80 ° C. in order to eliminate the phenomenon of stacking of the bases creating a disturbance of the hyperchromicity due to the existence of ternary structures We can therefore know the molecular extinction coefficients of the dodecamers a 80ºC according to the relation ε 265 (SrU ₁₂ ) = 12ε ²⁶⁵ (rU) B - Hybridization conditions.

Hybrid experiments are carried out with a UVIKON 810 spectrophotometer (KONTRON). coupled to a compatible IBM microcomputer. The temperature is controlled by a HUBER PD 415 temperature programmer connected to a thermostate bam (HUMER MINISTAT). The U .V. are made of quartz 1 cm long and a continuous circulation of nitrogen is used for temperatures below 20ºC Before the experiment. 3 μM (Examples I to V) or 10 μM (Examples VI and V II) of additional oligomers are brought into contact in a quantity sufficient for 1000 μl of 1 M ΝaCl buffer and 10 mM sodium cocodylate, adjusted to pH 7 ( Examples I to V II) This mixture is heated to 80 ° C for 1 hour, then cooled to -20 ° C according to a temperature slope of 0.5 ° C per minute. Absorbance and temperature values are collected every 30 seconds.

 The same experiment is carried out using a quantity sufficient for 1000 μl of 0.1 M ΝaCl hybridization buffer. 10 mM sodium cacodylate adjusted to pH - in order to evaluate the thermal subsistence of the duplex in this buffer which favors less pairings

EXAMPLE I Thermal Subility of the Duplex ßSrU ₁₂ / PolyrA

I-1 Determination of oligomers

SsSrU ₁₂ concentration = 0.351mM

PolyrA concentration = 4.61 mM

I-2 Hybridization conditions

3 μM (8.55 μl) of ßSrU ₁₂ and 3 μ.M (7.79 μl) of polyrA are placed in the presence of 983.6 μl of the dilution buffers (1 M ΝaCl. 10 mM sodium cacodylate or 0.1 M ΝaCl. 10 m, M cacodylate sodium)

Example II: Thermal Subility of the ßrU ₁₂ / PolyrA Duplex In progress

EXAMPLE III Thermal Subility of the Duplex ßSrU ₁₂ / ßdC ₂ A ₁₂ C ₂

III-1 Determination of oligomers

SsSrU ₁₂ concentration = 0.351 mM

Concentration of ßdC ₂ A ₁₂ C ₂ = 0.369 mM

III-2 Hybridization conditions

3 μM (8.55 μl) of ßSrU ₁₂ and 3 μM (8.1 1 μl) of ßdC ₂ A ₁₂ C ₂ are placed in the presence of 983.3 μl of the hybridization buffers (1 M NaCl. 10 mM sodium cocodylate or 0.1 M NaCl . 10 mM sodium cacodylate)

Example IV: Thermal Subility of the Duplex ßrU ₁₂ / ßdC _{2 to 12} C ₂ (in progress)

IV- 1 Determination of oligomers

Concentration of ßrU ₁₂ =

Concentration of ßdC ₂ A ₁₂ C ₂ = 0.369 mM

III-2 Hybridization conditions

3 μM (μl) of ßrU ₁₂ and 3 μM (8.1 1 μl) of ßdC ₂ A ₁₂ C ₂ are placed in the presence of μl of the hybridization buffers (1 M NaCl. 10 mM sodium cacodylate or 0.1 M NaCl. 10 mM sodium cacodylate)

Example V: Self-hybridization experiment of the ßSrLχ2 oligomer

V-1 Determination of oligomers

SsSrU ₁₂ concentration = 0.351 mM

V-2 Hybridization conditions

6 μ.M (17, 10 μl) of ßSrU ₁₂ are placed in the presence of 986.9 μl of the hybridization buffers (1 M NaCl. 10 mM sodium cacodylate or 0.1 M NaCl, 10 mM sodium cacodylate)

Example VI: Thermal Subility of the Duplex ßdT ₅ (SrT) dT ₆ / ßdC ₂ A ₁₂ C ₂

VI- 1 Determination of oligomers

Concentration of ßdT ₅ (SrT) dT ₆ = 1.33 mM

Concentration of ßdC ₂ A ₁₂ C ₂ = 0.369 mM

VI-2 Hybridization-conditions 10 μM (7.49 μl) of ßdT5 (SrT) dT ₆ and 10 μM (22.5 μl) of ßdC ₂ A ₁₂ C ₂ are placed in the presence of 970 μl of hybridization buffer 1M NaCl, 10 mM sodium cocodylate

Example VII: Thermal stability of the ßdT ₁₂ / ßdC duplex ₂ A ₁₂ C ₂ VII-1 Determination of the oligomers

SsdT ₁₂ concentration = 1.196 mM

SsdC ₂ concentration at ₁₂ C ₂ = 0.369 mM VII- 2 Hybridization conditions

10 μM (8.36 μl) of ßdT] 2 and 10 μM (22.5 μl) of ßdC ₂ A ₁₂ C ₂ are placed in the presence of 969 1 μl of 1 M NaCl hybridization buffer. 10 mM sodium cacodylate,

V. HYBRIDIZATION PROPERTIES OF 4'-S-RNA AND MIXED OLIGOMERS. Hybridization experiments are carried out while respecting the 1/1 stoichiometry between the two complementary strands in the presence of a 1 M NaCl buffer. 10 mM sodium cacodylate A high concentration of NaCl indeed promotes pairings by solvating the negative charges around the phosphorus atoms of the two oligonucleotides. We carried out these hybridization experiments in another 0.1 M NaCl buffer, 10 mM sodium cacodylate,

V-l-RESULTS AND DISCUSSION.

Duplex fusion experiences:

[4'-SrU ₃ -dT ₆ -4'-SrU ₃ / P ₁₁ ] / polyrA, [4 / polyrA]

[4'-SrU ₃ -dT ₆ -4'-SrU ₃ / P ₁₁ ] / d (C ₂ A ₁₂ C ₂ ), [4 / d (C ₂ A ₁₂ C ₂ )]

[4'-SrU ₃ -dT ₆ -4'-SrU ₃ / P ₃ PS ₅ P ₃ ] / polyrA [5 / polyrA]

[4'-SrU ₃ -dT ₆ -4'-SrU ₃ / P ₃ PS ₅ P ₃ ] / d (C ₂ A ₁₂ C ₂ ), [5 / d (C ₂ A ₁₂ C ₂ )]

are carried out at two NaCl concentrations and make it possible to evaluate the influence of the phosphorothioate bonds on the hybridization stability compared to the oligonucleotides with normal phosphodiester bonds (table 7).

REPLACEMENT SHEET

The mixed dodecamer 5 exhibiting both phosphodiester and phosphorothioate intemucleotide bonds (P ₃ PS ₅ P ₃ ) induces a decrease in the value of Tm of approximately 2 ° C. by phosphorothioate bond. On the other hand, the oligomer 4 has Tm values which are equivalent to those obtained for the dodecamer ßrSU ₁₂ with the two complementary sequences poly rA and d (C ₂ A ₁₂ C ₂ ). These results (Table 7) indicate that the oligomers of mixed sequence 4 and 5 exhibit good thermal subsistence with complementary RNA sequences.

EXPERIMENTAL PART.

General method for hybridization experiments

A - Determination of complementary oligomers.

In order to carry out the hybridization experiment, it is necessary to know the concentration of each oligomer in order to respect the 1: 1 stoichiometry between the two complementary strands According to the law of BEER-LAMBERT A = εlc. the absorbances of the oligomers were measured at 80 ° C. in order to eliminate the phenomenon of stacking of the bases creating a disturbance of the hyperchromicity due to the existence of tertiary structures. We can therefore know the molecular extinction coefficients of dodecamers at 80 ° C according to a relationship similar to ε ²⁶⁵ (SrU ₁₂ ) ~ 12 ε ²⁶⁵ (SrU).

B - Hybridization conditions.

Hybridization experiments are carried out with a UVIKON 810 spectrophotometer (KONTRON), coupled to an IBM compatible microcomputer. The temperature is controlled by a HUBER PD 415 temperature programmer connected to a thermosute bath (HUMER MINISTAT). The UV tanks are made of quartz 1 cm long and a circulation continual nitrogen is used for temperatures below 20 ° C. Before 1 experiment, 3 μM (Examples I to IV) of additional ohmomers are brought together in an amount sufficient for 1000 μl of 1 M NaCl buffer and 10 mM cacodylate of sodium adjusted to pH 7 (Examples I to VII) This mixture is heated to 80 ° C for 1 hour, then cooled to -20 ° C according to a temperature slope of 0.5 ° C per minute The absorbance values and temperature are collected every 30 seconds

The same experiment is carried out using a quantity sufficient for 1000 μl of 0.1 M NaCl hybridization buffer, 10 mM sodium cacodylate adjusted to pH 7 (Example I and IV) in order to evaluate the thermal subthte of the duplex in this buffer less favoring pairings

Example I: Thermal subtility of the duplex (4'-SrU ₃ -dT ₆ -4'-SrU ₃ / P ₁₁ ] / polyrA, 4 / polyrA.

I-1 Determination of oligomers

Concentration of 4 = 0.821 mM

PolyrA concentration = 4.61 mM

I-2 Hybridization conditions

 3 μM (3.65 μl) of 4 and 3 μM (7.79 μl) of polyrA are placed in the presence of 988 5 μl of the hybridization buffers (1 M NaCL 10 mM sodium cacodylate or 0.1 M NaCl 10 mM sodium cacodylate )

Example II: Thermal Subility of the Duplex 4'-SrU ₃ -dT ₆ -4'-SrL ₃ / P ₁₁ / dC ₂ A _{1 2} C ₂ , 4 / dC ₂ A ₁₂ C ₂ ll-1 Determination of Oligomers

Concentration of 4 = 0.821 mM

DC ₂ concentration at ₁₂ C ₂ = 0.369 mM

II-2 Hybridization conditions

3 μM (3.65 μl) of 4 and 3 μM (8.1 1 μl) of dC ₂ A ₁₂ C ₂ are placed in the presence of 988 2 μl of the hybridization buffers (1 M NaCl, 10 mM sodium cacodylate or 0.1 M NaCl , 10 mM sodium cacodylate)

Example III: Thermal Subility of the Duplex [4'-SrU ₃ -dT ₆ -4'-SrU ₃ / P ₃ PS ₅ P ₃ ] / PolyrA, 5 / PolyrA III- 1 Determination of Oligomers

 Concentration of 5 = 0.40 mM.

PolyrA concentration = 4.61 mM. III-2 Hybridization conditions

 3 μM (7.5 μl) of 5 and 3 μM (7.79 μl) of polyrA are placed in the presence of 984.7 μl of the hybridization buffers (1 M NaCl. 10 mM sodium cacodylate or 0.1 M NaCl. 10 mM sodium cacodylate)

EXAMPLE IV Thermal Subility of the Duplex

[4'-SrU ₃ -dT ₆ -4'-SrU ₃ / P ₃ PS ₅ P ₃ ] / dC ₂ A ₁₂ C ₂ , 5 / polyrA

IV-1 Determination of oligomers

Concentration of 5 = 0.40 mM

DC ₂ concentration at ₁₂ C ₂ = 0.369 mM

IV-2 Hybridization conditions

3 μM (7.5 μl) of 5 and 3 μM (8.1 1 μl) of dC ₂ A ₁₂ C ₂ are placed in the presence of 984.4 μl of the hybridization buffers (1 M NaCl. 10 mM sodium cocodylate or 0.1 M NaCl. 10 mM sodium cacodylate).

VI. ENZYMATIC STUDIES

The recognition of nucleic acids by enzymes is largely structural. The replacement of the natural heteroatom of the sugar of an oligonucleonde gives the latter a high resistance to degradation by nucleases.

 The study of the hydrolysis of 4′-thio-oligoribonucleotides ß by these nucleases makes it possible to confirm that the replacement of intracyclic oxygen by a sulfur atom does indeed confer better enzymic stability.

Example I: Study of the enzymatic degradation of the dodecamer ß (SrT) dT _{1 1} by calf spleen phosphodiesterase.

Calf spleen phosphodiesterase is an exonuclease which degrades oligomers from their free 5 'ends, thereby releasing nucleotide 3' phosphate.

This study will allow us to assess the resistance to enzymatic degradation induced by the presence in the 5 'position of a 4'-thionucleotide unit in the ß (SrT) dT _{1 1} oligomer compared to the enzymatic degradation of the natural counterpart. oxygenated ßdT ₁₂ The comparative subtility of the two dodecamers ßdT 12 and ß (SrT) dT _{1 1} towards hydrolysis by phosphodiesterase of calf spleen is presented in table 8. The difference in behavior between the two dodecamers is significant, in fact only 23% of the ß (SrT) dTn oϋgomer was hydrolyzed in 25 minutes while at the same time 12-mer ßdT 12 was degraded to 93 %.

Kinetics of degradation of ßdT ₁₂ and ß (SrT) dT _{1 1} by calf spleen phosphodiesterase

The evolution over time of the enzymatic degradation of the two dodecamers was followed by HPLC. We were able to plot the enzymatic digestion curve for ß (SrT.dT ι \ as a function of time: A = A ₀ e ^-kt (Figure 2. A) where:

- A ₀ is the initial aix of the 12-meter

 - A is the area of 12-mer at time t

 - t is the time

 - k is the first order rate constant of the degradation

This curve (Figure 2) allows us to calculate, using the curvilinear regression software EUREKA, k = 0.016min ^{- 1} and the half-life time of the dodecamer t _1/2 = In 2. k =

43 mins

In the same way, the enzymatic digestion curve of ßdT 12≈n as a function of time AA ₀ e ^-kt could be plotted. (Figure 2). It is seen that the degradation kinetics of ßdT ₁₂ is much faster than that of ß (SrT) dT _{1 1} ; indeed the half-life time is 1 minute for the natural dodecamer.

The kinetics of enzymatic degradation of the oligomer modified ß (SrT) dT ₁₁ by the 5 ′ end exhibits a half-life time clearly greater than that of the degradation of ßSrU ₁₂ . These results show that the introduction of a thioribonucleotide unit at the 5 ′ end of an oligomer subilizes the latter with respect to calf spleen phosphodiesterase.

An oligonucleotide solution (20 μl, 3 absorbance units at 260 nm) is diluted in 0.125 M ammonium acetate buffer (pH 7.0), 2.5 mM EDTA and Tween 80 0 0625% ( 80 μL). A commercial solution of calf spleen phosphodiesterase (2 μl. Final concentration 0.008 unit / ml) is added and the mixture is incubated at 37 ° C. for determined times, aliquots are taken (5 μl), heated at 100 ° C. for one minute and analyzed by HPLC.

 HPLC analysis conditions.

The analyzes were carried out on a PVDI column (Polyvinylimidazole SFCC-Shandon) protected by a guard column and a prefilter. Elution is carried out according to a linear gradient in 20 minutes from 0.1 M to 0.4 M KCl in 20 mM KH ₂ PO ₄ at pH 6 containing 20% acetonitrile. The flow rate was set at 1 ml / min and the UV detection at 260 nm,

EXAMPLE II Study of the substrate-type activities of hexamers 4'-SrU ₆ , 4'-SrU ₆ -3'n-prOH compared to the substrate characters of the hexamers βrU ₆ and αrU ₆ with respect to various purified nucleases.

Four typical nucleases were used:

 - The endonuclease S1, specific for the single strand of DNA, hydrolyzes the phosphodiester bonds in a random manner.

 - Calf spleen phosphodiesterase (CSP) is a 5'-exonuclease which degrades oligonucleotides from their 5 'end and releases nucleotide 3'-phosphates.

 - Snake venom phosphodiesterase (VSP) is a 3'-exonuclease-3 'which releases nucleotides 5'-phosphate after degrading the oligomer at its 3' end

 - Ribonuclease A degrades an oligorihonucleotide after pyrimidine residues

These four enzymes were obtained from Boehringer Mannheim,

In general, an optical density unit at 260 nm from each hexamer is placed in the presence of a certain concentration of enzyme considered then is diluted in an amount sufficient for 1000 μl of enzymatic digestion buffer.

 Different digestion buffers were used depending on the nature of the enzyme (Table 9).

The mother sample is divided into 10 fractions of 100 μl before being frozen at -18 ° C.

An aliquot fraction of 100 μl is taken and incubated in a dry thermostated oven at 37 ° C. for a time t and then is analyzed by analytical HPLC. The study of the signal area corresponding to the hexamer makes it possible to measure a degradation kinetics whose half-life time is calculated (Table 10).

The hexamer 4'-SrU _6, 2 has a high enzymatic resistance compared to that of ß rU ₆ against four nucleases (CSP. S ₁ , RNase-A and VSP).

Resistance to 3'-exonuclease is greatly increased by the introduction of a propanol phosphodiester bond on the 3 'end of 4'-SrU ₆ 3'n-prOH, 3.

CONCLUSION.

Studies have shown that: a) homogeneous or mixed 4'-thio-oligoribonucleotides bind with good thermodynamic stability with complementary RNA. b) the substitution of the cyclic oxygen by a sulfur atom induces a strong resistance to the enzymatic degradation (cf. the comparison of the half-life times of ß-rUß and ß-S- rU ₆ )

All of these data imply that 4'-thio-oligoribonucleotides can be considered as antisense agents in the form of homogeneous sequences or included in mixed oligomers. In addition, the presence of the 2 'hydroxyl function makes it possible to envisage their use as artificial ribozymes, whether in the form of homogeneous or mixed sequences associated with oligonucleotides of various DNA or RNA type, modified or not at the level of the chain. phosphate.

Claims

1. Chemical compounds constituted by an ohgo-4'-thio-2 'deoxyribonucleotide or an oligo 4'-thioribonucleoride, characterized in that they comprise a sequence of 4'-thio-2' deoxyribonucIeotides or 4'-thioribonucleotides respectively, chain which can optionally be linked to an effector agent, in particular a radical corresponding to an intercalating agent or a chemical or photoactivable radical, such as a radical carrying a function reacting directly or indirectly with the nucleotide chains or a radical of which presence allows easy and sensitive detection.

 2. Chemical compounds according to claim 1 consisting of a 4'-thioribonucleotide oligo or a 4'-thio-2'-deoxyribonucleotide oligo, characterized in that they comprise a sequence of 4-thioribonucleotides or 4-thio-2'- deoxyribonucleotides respectively not linked to an effector radical.

 3. Compounds according to claim 1 or 2 characterized in that they have the formula:

in which :

the β radicals may be the same or different and each represents a base of an optionally modified nucleic acid, activatable and / or comprising an intercalating group and attached to the glycosidic cycle according to an unnatural alpha anomeric configuration; the radicals X may be the same or different and each represents an oxoanion O -, a thioanion S -, an alkyl group, an aicoxy group, a thioalkyl group, an alkyl or alkoxy radical substituted by a nitrogen heterocycle or a group -YZ;

 R and R ', which may be the same or different, each represents a hydrogen atom or a group -Y-Z or Y'-Z';

Y and Y ', identical or different, each represent a straight or branched alkylene radical -alk- or a radical chosen from

with U = O, S or NE can have the same meanings as X except YZ or

Y'-Z ';

 J represents a hydrogen atom or a hydroxy group; Z and Z ', identical or different, each represent CH or a radical corresponding to an effector, in particular a radical corresponding to an intercalating agent or a radical carrying a function which reacts directly or indirectly with the nucleotide chains or a radical whose presence allows easy and sensitive detection.

 n is an integer including 0;

L represents an oxygen atom, a sulfur atom or a group -NH-.

4. Compounds according to claim 3, characterized in that Y and Y 'represent a radical chosen from

with U = O, 5 or N and n 'is an integer notably from 1 to 10.

 5. Compounds according to Claim 3, characterized in that L represents oxygen, R and R 'represent a hydrogen atom and ß represents a natural nucleic base.

 6. Compounds according to one of claims 3 to 5, characterized in that the intercalating agent Z or Z 'is chosen from polycychque compounds having a plane configuration.

 7. Compounds according to Claim 6, characterized in that the intercalating agent Z or Z 'is chosen from acridine and its derivatives, furocoumarin and its derivatives, daunomycin and the other derivatives of anthracycline, 1 , 10-phenanthroiine, phenanthridine and its derivatives, proflavme, porphyrins, dipyrido [1,2-a: 3'-d] imidazole derivatives, ellipticine or ellipticinium and their derivatives and diazapyrene and derivatives.

 8. Compounds according to one of claims 3 to 5, characterized in that the reactive chemical groups Z or Z 'are chosen from ethylene-diamine-tetraacetic acid, diethylenetriaminepentaacetic acid, porphyrins, 1,10- phenanthroline, 4-azido acetophenone, ethyleneimine, beta-chloroethylamine, psoralen and their derivatives.

9. Compounds according to one of claims 3 to 8, characterized in that the radical B is a radical corresponding to thymine, adenine; 2-aminoadenine and its substituted derivatives, cytosine, guanine and its substituted derivatives, uracil, bromo-5 uracil, azido-8 adenine, 7-deazaadenine, 7-deazaguanine, 4-azido -cytosine, 4-azido-thymine and derivatives of these bases comprising an intercalating group and / or a chemically or photochemically activatable group.

10. Compounds according to one of claims 1 to 9, characterized in that they are oligothioribonucleotide compounds of formula 1 for which J = OH,

 1 1. Mixed oligomer compounds characterized in that they consist of compounds according to one of claims 1 to 10 linked to oligonucleotides of DNA or RNA type modified or not at the phosphate chain and in which the heteroatom intracyclic of the furanosis cycle of nucleotides is oxygen.

 12. Mixed oiomeric compounds characterized in that they consist of a compound according to one of claims 1 to 10, in particular of formula (I), comprising, at one or at each of its ends, an oligonucleotide sequence of DNA type or RNA modified or not at the level of the phosphate chain, in which the heteroatom of the furanosis cycle of the nucleotides is oxygen.

 13. mixed oiomeric compounds according to claim 1 2 characterized in that they consist of an oligothioribonucleotide compound according to one of claims 1 to 10, comprising a DNA type sequence modified or not at the level of the phosphate chain in the nucleotides of which the heteroatom of the furanosis cycle of the nucleotides is oxygen.

 14. A mixed oligomer compound according to claim 13. characterized in that said DNA sequence, in which the heteroatom of the furanosis cycle is oxygen, is surrounded by two sequences of the thioribonucleotide type.

 15. Process for the preparation of compounds according to one of claims 1 to 13, characterized in that syntheses are applied to the phosphodiester, phosphotriester, phosphoramidite or hydrogenophosphonate using 4thionucieosides for oligothionucieotide sequences and in what is prepared totally protected derivatives and that the protective groups are eliminated.

16. Method for synthesizing compounds without an effective agent according to one of claims 2 to 1 3, characterized in that, in order to prepare the oligothionucieotide sequences, a supported synthesis is carried out according to the phospohoroamidite method comprising - 3 'and 5' protection of the starting 4'thionucleotides or oligo-4'thionucleotides with, for example, dimethoxytrityl at 5 'and methyl dnsopropylaminophosphoramidite at 3',

the functionalization of a solid support incorporating a 4'thionucleoside derivative by a link, for example succinyl, between the 3'-hydroxyl group of the 4'thionucleoside derivative and an amino group of the solid support,

 - elongation of the oligothionucleotide chain in a synthesizer reactor,

 - finally the stall, deprotection and purification of the extended oligothionucleotide.

 17. Process for the synthesis of compounds incorporating an effector agent according to one of claims 3 to 10, characterized in that one starts from an oligothionucieotide without effector agent, or mixed ohgomer comprising, protected in 5 ', of which reacts the OH 3 ′ termination with an unprotected function of a difunctional arm whose second function is protected and after deprotection of the resulting product, said product is linked to the effector by the second free function of the difunctional arm.

 18. Application of the compounds according to one of claims 1 to 13, characterized in that said compounds are used as hybridization probes or as purification components for specific DNA or RNA sequences in single strand or double helix for diagnostic or prognostic purposes.

 19. Application of the compounds according to one of claims 1 to 13, characterized in that the compounds are used to detect mutations in a DNA or an RNA.

 20. Application of the compounds according to one of claims 1 to 13, characterized in that these compounds are used to achieve the specific blocking of cellular genes or pathogens previously chosen, such as viruses, bacteria or parasites.

21. Application of a derivative according to one of claims 1 to 13 to selectively block the expression of a gene either by hybridization. either by selective cutting, or by chemical modification of the gene or its messenger RNA of cellular, viral, bacterial or parasitic origin.

22. Application of a derivative according to one of claims 1 to 1 3 for selectively blocking the expression of a viral RNA either by hybridization, or by cleavage, or by chemical modification of the viral RNA or of its messenger RNA .

 23. Application of the compounds according to one of claims 1 to 13, characterized in that they can be used as specific artificial nucleases rde sequences or artificial ribozyme.

 24. As medicaments, compounds according to one of claims 1 to 13, usable as antiviral, antitumor, antibiotic or antiparasitic or in any pathology where a gene is abnormally expressed either by mutation or by deregulation.